Compositions and Methods for the Intracellular Disruption of VEGF and VEGFR-2 by Intraceptors

ABSTRACT

The present invention provides an intraceptor that interacts with and decreases activity of with VEGF and/or a VEGFR for the treatment of angiogenesis-related conditions. The present invention further provides pharmaceutical compositions, and methods of use thereof for the treatment and prevention of an angiogenesis-related condition using said intraceptors. The invention further provides for nucleic acids encoding said intraceptors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/231,629, filed on Mar. 31, 2014, which is a continuation of U.S.patent application Ser. No. 13/540,495, filed Jul. 2, 2012, which is acontinuation of U.S. patent application Ser. No. 11/814,890, filed Oct.22, 2007, which is the U.S. national stage entry of Patent CooperationTreaty Application No. PCT/US2006/002684, filed on Jan. 6, 2006, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/647,224 filed Jan. 26, 2005, each of which are hereby incorporated byreference.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates generally to compositions and methods forinhibiting angiogenesis. In particular, this invention relates tocompositions comprising intraceptors, and methods of use thereof, todisrupt the intracellular expression and/or secretion of a vascularendothelial growth, factors (VEGF) and/or vascular endothelial growthfactor receptor (VEGFR). In certain embodiments, such intraceptors canbe used to treat angiogenesis related condition.

Background Art

Angiogenesis, the growth of new blood vessels, is a fundamentalbiological process which plays a central role in the pathogenesis ofvarious conditions, and is a major contributor to mortality andmorbidity in diseases, such as cancer, diabetic retinopathy, and maculardegeneration (Folkman, 1990, JNCI 82; 4-6). Cancer is the second loadingcause of death in the United States, claiming 553,251 lives in 2001(National Vital Statistics Report. 2003). Diabetic retinopathy affectsover 53 million Americans, and is the leading cause of new blindnessamong U.S. adults 20-74 years of age. Proliferative diabetic retinopathy(PDR) is a condition in which, abnormal new blood vessels in the retinamay rupture and bleed inside the eye, and is a principal cause ofblindness in diabetics. The prevalence of PDR increases from 2% atdiagnosis to 20% after 20 years of disease (Morbidity and MortalityWeekly Report, 1993, pp. 191-95). The estimated incidence of new PDRcases is about 65,000 per year. Age-related macular degeneration (AMD)is the leading cause of irreversible blindness among those over 65 inthe United States, Western Europe, and Japan and affects over 11 millionpersons in the US. More than 20% of the American population is olderthan 55 years of age and at risk for AMD. Each year, more than a millionindividuals softer severe central vision loss due to AMD; these numberswill, skyrocket with the aging population. Neovascular AMD isresponsible for severe vision loss in 80-90% of these patients (MacularPhotocoagulation Study Group. Arch Ophthalmol. 1991; 109:1109-14).

Corneal neovascularization is a central feature in the pathogenesis ofmany blinding corneal disorders, and a major sight-threateningcomplication in corneal infections, chemical injury, and following,keratoplasty, in which neovascularization adversely impacts cornealtransplant survival (Epstein et al., 1987, Cornea. 6:250-57).Anti-angiogenic molecules have been shown to inhibit cornealneovascularization (Ambati et al., 2002, Arch Ophthalmol 120; 1063-68).Thermal laser and photodynamic therapy induces only temporary closure ofnew vessels (Primbs et al., 1998, 29; 832-38), without addressing theunderlying biology of neovascularization.

Annually, approximately 45,000 corneal transplants are performed in theUS. This is the highest number for any transplant, largely because ofhigh success conferred by the immune privilege of the normally avascularcornea. Corneal neovascularization breaches this immune privilege, andis a major factor in rejection of corneal transplants, which occurs inabout 10% of cases.

Vascular endothelial, growth factor (VEGF) is a key mediator ofangiogenesis in many models (Nenfeld et al., 1999, FASEB J. 13; 9;Dvorak, 1999, Curr. Top Microbiol. Immunol. 237: 97; and Carmeliet &Collen, 1999, Curr. Top Microbiol. Immunol. 237: 133, etc.). VEGFpromotes vascular endothelial cell migration, proliferation, inhibitionof apoptosis, vasodilation, and increased vascular permeability. Inseveral clinically relevant models of animal and human cornealneovascularization, angiogenesis is driven by increased secretion ofVEGF-A (herein referred to as VEGF) (Amano et al., 1998, InvestOphthalmol Vis Sci. 18-22; Cursiefen et al., 2004, J. Clin Invest. 113:1040-50; and Philipp et al., 2000, Invest Ophthalmol Vis Sci. 41:2514-22), and is also closely linked to infiltrating leukocytes (Amanoet al., 1998, Invest Ophthalmol Vis Sci. 18-22).

Three receptors constitute the VEGF receptor family, which includesVEGFR-1 (Flt or Flt-1), VEGFR-2 (KDR), and VEGFR-3 (Flt-4), all of whichhave tyrosine-kinase activity (Neufeld et al., 1999, FASEB J. 13.9). ThecDNA and amino acid sequences of human Flt-1 are found at accessionnumber gi:56385329. Several studies have shown VEGFR-2 (throughactivation of MAP kinase and P1-3K (phosphatidylinositol 3-kinase) isthe signal transducer tot VEGF-induced mitogenesis, chemotaxis, andcytoskeletal reorganization and thus the principal receptor involved inangiogenesis (Thakker et al., 1999 J. Biol. Chem. 274; 10002-7; Dimmeleret al., 2000, FEBS Lett 477:258-62; Carmeliet & Collen, 1999, Curr. TopMicrobol. Immunol. 237:97; Neufeld et al., 1999% FASEB J. 13:9; andMillauer et al., 1993, Cell 72: 835-46). VEGFR-3 is primarily involvedin lymphangiogenesis (Cursiefen et al., 2004, Invest Ophthalmol Vis Sci.45; 2666-73; Cursiefen et al., 2004, J. Clin Invest 113: 1040-50).

VEGF transcription is amplified in response to oncogenes, hypoxia, andother insults. Transcription factors for VEGF (HIF-1α and HIF-2α) arestabilised during hypoxia (Ahmed et al., 2000, Placenta 21 SA:S16-24;Wenger & Gassman, 1997, Biol. Chem. 378:609). Sensitivity to hypoxia isa major difference between VEGF and other angiogenic factors (Arbiser etal., 1997, Proc. Natl. Acad. Sci USA 94: 861; Okada et al., 1998, Proc.,Natl, Acad, Sci. USA 95: 3609; and Petit at al, 1997, Am. J, Pathol.151:1523). Elevated VEGF has been associated with a poor prognosis incancer and with diabetic retinopathy (Ambati et al., 1997, ArchOphthalmol 115: 1161-66). Strategies to inhibit VEGF have includedblocking antibodies, decoy receptors for VEGF, and anti-VEGF antibodies(Kim et al., 1993, Nature 362; 841-44; Yuan, et al., 1996, Proc. Natl.Acad, Sci, USA. 93:14765-70; Lin et al, 1998, Cell Growth Differ. 9:49-58; and Hasan & Jayson, 2001, Expert Opin Biol Ther. 1: 703-18).These strategies have generally reduced neovascularization by only30-50% (Robinson et al., 1996, Proc. Natl Acad Sci USA 93; 4851-56;Aiello et al. 1995, Free, Natl. Acad. Sci USA 92: 10457-61; Shen et al.,2002, Lab Invest 82:167-82; and Honda et al., 2000, Gene Ther.7:978-75). These levels of neovascularization reduction are insufficientfor the cornea, where angiogenesis should be minimized as much aspossible for optimal visual clarity.

Further, in the course of normal VEGF signal transduction, membrane Fltheterodimerizes with VEGFR-2 upon VEGF binding (Autiero et al., 2003,Nat. Med.; and Kendall et al., 1996, Biochem Biophy Res Comm.226:324-28). Physiologic Flt/VEGFR-2 heterodimers stimulate expressionof fee genes for the transcription factor Ets-1 and matrixmetalloproteinase 1 (MMP-1), phosphorylation of focal adhesive kinase(FAK), vinculin assembly and DNA synthesis (Kanno et al., 2000, Oncogene19: 2138-46; Sato et al., 2000, Ann NY Acad Sci. 902:201-7). Ets-1induces expression of Beatrix metalloproteinase 1 (MMP-1), MMP-3, MMP-9,matrix plasminogen activator; and β3 integrin b, all involved, inmatrix-neovessel interactions. MMP-1 facilitates digestion, ofextracellular matrix to facilitate vascular ingrowth, while FAK helpsmediate adhesion among endothelial cells and extracellular matrix. Theseevents are critical to endothelial cell migration and proliferation.

KDEL (SEQ ID NO: 1) is the one letter sequence for the peptide retentionsignal having the amino mid sequence Lys-Asp-Glu-Leu (SEQ ID NO: 1)which, binds endoplasmic reticulum retention receptors, thus preventingsecretion of ligands of proteins coupled to the sequence (Pelham, 1990,Trends Biochem Sci. 15:483-6). This is also a highly specific retentionsequence, as constructs using a KDEV (SEQ ID NO:2) sequence are notsuccessful at retaining targets (Tang et al. 1992, J. Biol Chem.267:7072-6), Although the mechanism of clearance or degradation ofKDEL-sequestered (SEQ ID NO:1) proteins is not fully known, theubiquitin-proteasome pathway is thought to be the principal route forclearance of intraceptor-retained proteins, as removal of KDEL (SEQ IDNO:1) from PDI, an ER chaperone has recently been described to releaseits target protein, procollagen 1, from ubiquitin-proteasome degradation(ko & Kay, 2004, Exp Cell Res. 295: 25-35).

Linkage of KDEL (SEQ ID NO:1) to chemokines (known as creation of“intrakines”) downregulates cognate receptors with significant roles indisease (Chen et al., 1997, Nat. Med. 3: 1110-6; Kreitean et al., 1995,Cancer Res. 55: 3357-63). Coupling stromal derived factor (SDF) wifeKDEL (SEQ ID NO: 1) blocked cell surface expression of SDF's receptor,CXCR-4; similar efforts have been used to downregulate cell surfaceexpression of other receptors, including CCR-5 and Interleukin-4receptor (Kreitman et al., 1995, Cancer Res. 55: 3357-63; Luis et al.,2003, Mol Ther. 8: 475-84; and Steinberger et al., 2000, Proc Natl Acad.Sci. 97: 805-10).

It has been, reported that sequestered proteins are eventually degradedin the endoplasmic reticulum (Pelham, 1990, Trends Biochem Sci. 15:483-6). The accumulation of sequestered, proteins in the endoplasmicreticulum may lead to endoplasmic reticulum overload, triggering theunfolded protein response (UPR), which could cause apoptosis ofendothelial cells, as the presence of unfolded proteins in endoplasmicreticulum (ER) leads to a stress response including release ofpro-apoptotic factors such as CHOP and caspase-12 (Wang et al., 1998,EMBO J. 17: 5708-17; Yoshida et al., 2001, Cell 107: 881-91; Tirasophonet al., 1998, Genes Dev. 12:812-24; Fornace et al., 1988, Proc Natl AcadSci USA 85: 8800-4; Kaufman, 2002, J Clin Invest 110: 1389-98; andSchroder & Kaufman, 2005, Mutat Res. 569; 29-63). Although the effect ofKDEL (SEQ ID NO:1)-mediated protein retention on these molecularresponses is unknown, it has been reported that the KDEL (SEQ ID NO:1)receptor is involved in the ER stress response (Yamamoto et al. 2003, J.Biol Chem. 278: 34525-32).

VEGF is an important target for inhibiting angiogenesis. Molecularinterventions such as anti-VEGF aptamers or antibodies (e.g. Macugen;Eyetech) and ranibizumab (Lucentis; Genentech) are currently used orunder investigation for AMD and PDR, but are based on extracellularblockade of VEGF. Intracellular approaches against VEGF couldpotentially ameliorate these conditions, as results for extracellularmodalities have been mixed (Gragoudas et al., 2004, N Engl J Med 351;2805-16), or add a new additional means to affectively reduce total VEGFfunction.

It is important to target VEDF intracellularly, as several cell typesrespond to their own VEGF production in an autocrine fashion. VEGFautocrine loops have also been demonstrated in endothelial cells (Hondaet al, .2000, Gene Ther. 7: 978-75; Lee et al, 1999, Eur J Cancer 35:1089-93), including in hypoxic HUVEC cells (Lee et al, 1999, Eur JCancer 35: 1089-93; Liu & Ellis, 1998, Pathobiology 66; 247-52);further, VEGF can upregulate its own receptor VEGFR-2 (Shen et al.,1998, J. Biol Chem. 273: 29979-85). Cancer cells producing VEGF andVEGFR-2 include prostate carcinoma, leukemia, pancreatic carcinoma,melanoma, Kaposi's sarcoma, and osteosarcoma (Lee et al. 1999, Eur JCancer 35: 1089-93; Masood et al., 2001, Blood 98: 1904-13).Intracellular autocrine loops render cells resistant to modalitiestargeting VEGF extracellularly (Gerber et al., 2002, Nature 417: 954-58;Santos & Dias, 2004, Blood 103: 3883-9).

Intracellularly disrupting VEGF expression is potentially superior toextracellular blockade by antibodies or aptamers as intracellular genesilencing may sabotage intracellular autocrine loops that have beendemonstrated for VEGF in cancer and endothelial cells (Lee et al., 1999,Eur J Cancer. 35: 1089-93; Liu & Ellis, 1998, Pathobiology 66: 247-52;Casella et al., 2003, Blood 101: 3316-23; Gerber et al. 2002, Nature417; 954-58; Straume & Akslen, 2001, Pathol. 159: 223-35). Intracellulardisruption, of VEGF signaling may represent a powerful addition to theanti-angiogenic arsenal, by sabotaging VEGF secretion and intracellularautocrine loops.

Alternative gene silencing approaches allying on RNAi, antisenseoligonucleotides or ribozymes for disrupting VEGF expression, andapproaches to sequester VEGF using PIGF-KDEL (SEQ ID NO:1) arepreviously described in the art. However, since placental growth factor(PIGF) can heterodimerize with VEGF or a complex of an anti-VEGF Fabfragment with KDEL (SEQ ID NO:1; Wheeler et al., 2003, FASEB J 17:1733-5), there is a need to develop a more effective approach fordisrupting both VEGF and VEGFR-2 intracellularly for treating orpreventing angiogenesis. Such approach can induce the unfolded proteinresponse in cells that produce VEGF, resulting in selective ER stress.Furthermore, such approach is able to disrupt physiologic heterodimerformation of Flt/VEGFR-2, providing high specificity and affinity forthe target molecule due to the use of a receptor as the therapeuticsubstance.

SUMMARY OF THE INVENTION

The present invention provides for an intraceptor comprising apolypeptide encoded by a nucleotide sequence encoding at least a portionof an extracellular receptor, operatively linked to a signal retentionpeptide. In certain embodiments, the intraceptor interacts with a ligandfor said extracellular receptor, and interaction decreases activity ofthe extracellular receptor. The invention also encompasses an isolatednucleic acid encoding at least a portion of an extracellular receptor,operatively linked to a nucleic acid encoding a signal retentionpolypeptide.

In specific embodiments, the invention contemplates an isolated nucleicacid comprising a mil-length polynucleotide selected from the groupconsisting of (a) a polynucleotide as defined in SEQ ID NO:3; (b) apolynucleotide as defined in SEQ ID NO:S; (c) a polynucleotide encodinga polypeptide as defined in SEQ ID NO:4; (d) a polynucleotide encoding apolypeptide as defined in SEQ ID NO: 6; and (e) a polynucleotidecomplementary to a full-length polynucleotide of any one of a) throughd) above. Also contemplated are an isolated nucleic acid comprising afull-length polynucleotide encoding a polypeptide having at least 80%sequence identity with a polypeptide as defined in SEQ ID NO:4 or SEQ IDNO:6, and wherein said polypeptide Interacts with VEGF and/or VEGFR-2,and an isolated nucleic acid comprising a polynucleotide that hybridizesunder highly stringent conditions to a second nucleic acid selected fromthe group consisting of (a) a polynucleotide as defined in SEQ ID NO:3or SEQ ID NO:5; and (b) a polynucleotide encoding a polypeptide asdefined in SEQ ID NO: 4 or SEQ ID NO:6, wherein said nucleic acidencodes a polypeptide that interacts with VEGF and/or VEGFR-2, andwherein the stringent conditions comprise hybridization in a 6×SSCsolution at 65° C. It is contemplated that the interaction of theencoded polypeptide with VEGF and/or VEGFR-2 will decrease the activityof VEGF and/or VEGFR-2.

The invention also encompasses specific intraceptors, such as anintraceptor that interacts with VEGF and/or a VEGFR, comprising achimeric polypeptide comprising a portion of SEQ ID NO: 13 operativelylinked to a signal retention peptide or a polypeptide having at least80% sequence identity with 30 consecutive amino acids of SEQ ID NO: 13operatively linked to a signal retention peptide. In certainembodiments, the portion, of SEQ ID NO: 13 comprises amino acids 1-305of SEQ ID NO:6; or comprises amino acids 1-211 of SEQ ID NO:4. In otherembodiments, the intraceptor is selected from the group consisting of(a) a polypeptide as defined in SEQ ID NO:4; (b) a polypeptide asdefined in SEQ ID NO: 6; and (c) a polypeptide having at least 80%sequence identity with the polypeptide of a) through b) above. In otherembodiments, the chimeric polypeptide comprises a polypeptide having atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence Identity with at least 30,40, 50, 60, 70, 80, 90, 100 or more consecutive amino acids of SEQ IDNO: 13 operatively linked to a signal retention peptide.

It is contemplated that the VEGFR can be selected from the groupconsisting of VEGFR-1, VEGFR-2 and VEGFR-3. In one embodiment, the VEGFRis VEGFR-2.

In certain embodiments, said signal retention peptide can preventsecretion of a peptide operatively linked to the signal retentionpeptide. In one embodiment, the signal retention peptide is anendoplasmic reticulum signal retention peptide selected from the groupconsisting of SEQ ID NOs:1, 7, or 8. In particular embodiments, theendoplasmic reticulum signal retention peptide is SEQ ID NO:1.

The invention also contemplates a pharmaceutical composition fortreating an angiogenesis-related condition comprising an intraceptor.Also encompassed within the invention are methods of heating anangiogenesis-related condition using the intraceptors and pharmaceuticalcompositions described herein to contact a cell involved in theangiogenesis-related condition. Preferably, the angiogenesis-relatedcondition is selected from the group consisting of inflammation, stroke,hemangioma, solid tumors, leukemias, lymphomas, myelomas, metastasis,telangiectasia psoriasis scleroderma, pyogenic granuloma, myocardialangiogenesis, plaque neovascularization, coronary collaterals, ischemiclimb angiogenesis, corneal diseases, robeosis, neovascular glaucoma,diabetic retinopathy, retrolental fibroplasia, arthritis, diabeticneovascularization, macular degeneration, wound healing, peptic ulcer,fractures, keloids, vasculogenesis, hematopoiesis, ovulation,menstruation, placentation, polycystic ovary syndrome, dysfunctionaluterine bleeding, endometrial hyperplasia and carcinoma, endometriosis,failed implantation and subnormal foetal grow, myometrial fibroids(uterine leiomyomas) and adenomyosis, ovarian hyperstimulation syndrome,ovarian carcinoma, melanoma, venous ulcers, acne, rosacea, warts,eczema, neurofibromatosis, tuberous sclerosis, and chronic inflammatorydisease. More preferably, the angiogenesis-related condition is selectedfrom the group consisting of melanoma, diabetic retinopathy, and maculardegeneration.

The invention further encompasses a method of inhibiting angiogenesis ina biological sample, comprising (a) providing a biological sample; and(b) combining the sample with a angiogenesis-inhibiting amount of anintraceptor that decreases activity of VEGF and/or a VEGF-R, whereincomprises a chimeric polypeptide comprising a portion of SEQ ID NO: 13operatively linked to a signal retention peptide, or a polypeptidehaving at least 80% sequence identity with 30 consecutive amino acids ofSEQ ID NO: 13 operatively linked to a signal retention peptide, whereinsaid contact decreases activity of VEGF and/or a VEGFR. It iscontemplated that the biological sample can from a mammal, orspecifically, can be a human biological sample. It is farthercontemplated that the biological sample can be in a patient and thepatient have an angiogenesis-related condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents VEGFR-1 (FLT). It consists of 7 domains, and has thehighest affinity to VEGF, with a ten-fold higher binding affinity thanVEGFR-2. It is a tyrosine kinase receptor with seven Ig-likeextracellular domains and a tyrosine kinase domain with a long kinaseinsert. Domain deletion studies have shown that a subunit construct ofdomains 2-3 binds VEGF with near wild-type affinity and that domain 1serves as a secretion signal sequence. Further, domain 4 is necessaryfor receptor homodimerization of FLT and is believed to be necessary forthe observed heterodimerization of FLT and VEGFR-2.

FIGS. 2A-2C are schematics showing VEGF and cell membrane FLT (A)binding (B), and then heterodimerizing with cell membrane VEGFR-2 (C);the complex then activates transcription factors intracellularly.

FIGS. 2D and 2E are schematics illustrating cells expressing VEGF thatare transfected to express Flt23K (SEQ ID NO:4) or Flt24K (bothcontaining K.DEL(SEQ ID NO: 1) as a C-terminal tag). This will bin VEGFbefore it is released (D) and prevent VEGF secretion. Further, in cellsthat also express VEGFR-2, Flt24K will then heterodimerize with VEGFR-2as well (2E). The KDEL (SEQ ID NO:1) tag will ensure retention andultimate degradation within the endoplasmic reticulum.

FIG. 3A shows that VEGF in hypoxic HCE cells is decreased by thepresence of Flt23K (SEQ ID NO:4) and Flt24K.

FIG. 3B shows a Western blot analysis of human corneal epithelial celllysates. Blot analysis (anti-VEGF; 1:200) of HCEC transfected withpCMC.Flt23K (lane 1), Flt24K (lane 2), or control (lane 3). Only thecontrol lane displayed free VEGF. Lane MW contains a standard molecularweight ladder. The beta-actin internal control was equivalent in alllanes (blots not shown).

FIGS. 4A-4C illustrate that injury-induced corneal neovascularization issignificantly inhibited by the intraceptors of the present invention.FIG. 4A shows mouse corneas injected with saline, empty pCMV,pCMV.Flt23K, and pCMV.FIt24K, respectively, 1 week post corneal injury.Diffuse neovascularization is present in control, whileneovascularization is inhibited where intraceptors were injected. FIG.4B illustrates that both intraceptors suppress corneal VEGFconcentration 2 days after injury. FIG. 4C illustrates that delivery ofpCMV.Flt23K or pCMV.Flt24K significantly suppress corneal leukocyteinfiltration 1 week after injury relative to Sham saline/PBS or emptypCMV control.

FIG. 5A shows a Western blot (anti-VEGF antibody) of HMMC cellstransfected with intraceptors, grown in hypoxia 48 hours, andimmunoprecipitated with anti-FLT antibody.

FIG. 5B shows a Western blot (anti-VEGFR-2 antibody) of HMMC cellstransfected with, intraceptors, grows, in hypoxia 48 hours, andimmunoprecipitated with anti-FLT antibody. Control cells (transfectedwith empty pCMV vector) did not show bands for VEGFR-2 or VEGF followingimmunoprecipitation for FLT (blots not shown). Lane 1: pCMV.Flt24K; Lane2: pCMV.Flt23K.

FIG. 6 illustrates that pCMV.FIt23K significantly suppresses VEGFsecretion induced by hypoxia in HMMC (experiments done in triplicates;p<0.001; no significant difference in baseline VEGF levels or cellnumbers).

FIG. 7A illustrates that six weeks alter tumor implantation and weeklyinjections of either PBS or pCMV.Flt24K, HMMX xenograft sixe in nudemice was 632.8 cubic mm in control mice and 102.4 cubic mm in treatedmice.

FIGS. 7B and 7C show representative photographs of a melanoma in acontrol mouse and treated, mouse, respectively.

FIG. 8 shows a Western blot with anti-XBP-1 of hypoxic HMEC cellsshowing that intraceptors of the present invention upregulate splicedXBP-1 (MW of visible band in land 3 and 4 is about 55 kD). Beta-actininternal control was equivalent in all lanes (blots not shown) . . .Lane 1=PBS; 2=empty pCMV; 3=pCMV.Flt23K; 4=pCMV.Flt24K.

FIG. 9 shows a Western blot showing expression of the 30 kDa form ofXBP-1 in control corneas, and elevation of the active 55 kDa form incorneas injected with plasmids expressing the intraceptors of thepresent invention. Lane A shows mouse corneas injected, with empty pCMVvector; Lane B shows mouse corneas injected with pCMV.Flt23K; and Lane Cshows mouse corneas injected with pCMV.Flt24K,

FIG. 10 illustrates RT-PCT results for CHOP that was performed 24 hoursafter transfection of HMEC. The CHOP mRNA transcript as measured bysemiquantitative densitometry was elevated 60% by Flt23K and 80% byFlt24K over the levels in cells transfected with the control plasmid.Lane 1; HMEC cells in media; 2: transfected with empty pCMV; 3:transfected with pCMV.Flt23K; 4: transfected with pCMV.Flt24K.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods ate disclosed and described, it isto be understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood, thatthe terminology used herein is for fee purpose of describing specificembodiments only and is not intended to be limiting. It is further to beunderstood that unless specifically defined herein, the terminology usedherein is to be given its traditional meaning as known in the relativeart.

The present invention provides for an intraceptor comprising apolypeptide encoded by a nucleotide sequence encoding at least a portionof an extracellular receptor, operatively linked to a signal retentionpeptide. In certain embodiments, the intraceptor interacts with a ligandtor said extracellular receptor, and interaction decreases activity ofthe extracellular receptor. The invention also encompasses an isolatednucleic acid encoding at least a portion of an extracellular receptor,operatively linked to a nucleic, acid encoding a signal retentionpolypeptide.

In specific embodiments, the invention contemplates an isolated nucleicacid, comprising a full-length polynucleotide selected from the groupconsisting of (a) a polynucleotide as defined in SEQ ID NO:3; (b) apolynucleotide as defined in SEQ ID NO:5; (c) a polynucleotide encodinga polypeptide as defined in SEQ ID NO:4; (d) a polynucleotide encoding apolypeptide as defined in SEQ ID NO:6; and (e) a polynucleotidecomplementary to a full-length polynucleotide of any one of a) throughd) above. Also contemplated are an isolated nucleic acid comprising afull-length polynucleotide encoding a polypeptide having at least 80%sequence identity with a polypeptide as defined in SEQ ID NO:4 or SEQ IDNO:6, and wherein said polypeptide interacts with VEGF and/or VEGFR-2,and an isolated nucleic acid comprising a polynucleotide that hybridizesunder highly stringent conditions to a second nucleic acid selected fromthe group consisting of (a) a polynucleotide as defined in SEQ ID NO: 3or SEQ ID NO:5; and (b) a polynucleotide encoding a polypeptide asdefined in SEQ ID NO:4 or SEQ ID NO;6, wherein said nucleic acid encodesa polypeptide that interacts with VEGF and/or VEGFR-2, and wherein thestringent conditions comprise hybridation in a 6×SSC solution at 65° C.It is contemplated that the interaction, of the encoded polypeptide withVEGF and/or VEGFR-2 will decrease the activity of VEGF and/or VEGFR-2.

The invention also encompasses specific intraceptors, such as anintraceptor that interacts with VEGF and/or a VEGFR, comprising achimeric polypeptide comprising a portion of SEQ ID NO: 13 operativelylinked to a signal retention peptide or a polypeptide having at least80% sequence identity with 30 consecutive amino acids of SEQ ID NO: 13operatively linked to a signal retention peptide. In certainembodiments, the portion of SEQ ID NO: 13 comprises amino acids 1-305 ofSEQ ID NO:6; or comprises amino acids 1-211 of SEQ ID NO:4. In otherembodiments, the intraceptor is selected from, the group consisting of(a) a polypeptide as defined In SEQ ID NO:4; (b) a polypeptide asdefined in SEQ ID NO:6; and (c) a polypeptide having at least 80%sequence identity with the polypeptide of a) through b) above. In otherembodiments, the chimeric polypeptide comprises a polypeptide having atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 93%, 96%, 97%, 9.8%, 99% or more sequence identity with at least30, 40, 50, 60, 70, 80, 90, 100 or more consecutive amino acids of SEQID NO: 13 operatively linked to a signal retention peptide.

It is contemplated that the VEGFR can he selected from the groupconsisting of VEGFR-1, VEGFR-2 and VEGFR-3. In one embodiment, the VEGFRis VEGFR-2.

In certain embodiments, said signal retention peptide can preventsecretion of a peptide operatively linked to the signal retentionpeptide. In one embodiment the signal retention peptide is anendoplasmic reticulum signal retention peptide selected from the groupconsisting of SEQ ID NOs: 1, 7 or 8. In particular embodiments, theendoplasmic reticulum signal retention peptide is SEQ ID NO:1

The invention also contemplates a pharmaceutical composition fortreating an angiogenesis-related condition comprising an intraceptor.Also encompassed within the invention are methods of treating anangiogenesis-related condition using the intraceptors and pharmaceuticalcompositions described herein to contact a cell involved in theangiogenesis-related condition. Preferably, the angiogenesis-relatedcondition is selected from fee group consisting of inflammation, stroke,hemangioma, solid tumors, leukemias, lymphomas, myelomas, metastasis,telangiectasia psoriasis scleroderma, pyogenic granuloma, myocardialangiogenesis, plaque neovascularization, coronary collaterals, ischemiclimb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma,diabetic retinopathy, retrolental fibroplasia, arthritis, diabeticneovascularization, macular degeneration, wound healing, peptic ulcer,fractures, keloids, vasculogenesis, hematopoiesis ovulation,menstruation, placentation, polycystic ovary syndrome, dysfunctionaluterine bleeding, endometrial hyperplasia and carcinoma, endometriosis,failed implantation and subnormal foetal growth, myometrial fibroids(uterine leiomyomas) and adenomyosis, ovarian hyperstimulation syndrome,ovarian carcinoma, melanoma, venous ulcers, acne, rosacea, warts,eczema, neurofibromatosis, tuberous sclerosis, and chronic infammatorydisease. More preferably, the angiogenesis-related condition is selectedfrom the group consisting of melanoma, diabetic retinopathy, and maculardegeneration.

The invention farmer encompasses a method of inhibiting angiogenesis ina biological sample, comprising (a) providing a biological sample; and(b) combining the sample with a angiogenesis-inhibiting amount of anintraceptor that decreases activity of VEGF and/or a VEGF-R, whereincomprises a chimeric polypeptide comprising a portion of SEQ ID NO: 13operatively linked to a signal retention peptide, or a polypeptidehaving at least 80% sequence identity with 30 consecutive amino acids ofSEQ ID NO: 13 operatively linked to a signal retention peptide, whereinsaid contact decreases activity of VEGF and/or a VEGFR. It iscontemplated that the biological sample can from a mammal, orspecifically, can be a human biological sample. It is furthercontemplated that the biological sample can be in a patient and thepatient have an angiogenesis-related condition.

As used herein, VEGF is an abbreviation for Vascular Endothelial GrowthFactor, and VEGFR is an abbreviation for Vascular Endothelial GrowthFactor Receptor. There are three types of VEGFR, namely, VEGFR-1 (alsoknown as Flt), VEGFR-2 (also known as KDR or Flt), and VEGFR-3 (alsoknown as Flt-4), all of which have tyrosine-kinase activity.

As used herein, the term “signal retention peptide” or “peptideretention Signal” refers to an amino acid sequence that binds toretention receptors to prevent secretion of ligands of proteins coupledto such signal retention peptide. In one embodiment, the signalretention peptide binds endoplasmic reticulum retention receptors. Infurther embodiments, the signal retention peptide of the presentinvention binds endoplasmic reticulum retention receptors, preventingsecretion of a ligand that binds to a receptor. In additionalembodiments, the ligand that binds to a receptor is itself at least aportion of a receptor; such chimeric polypeptides comprising a signalretention peptide and at least a portion of a receptor are referred toherein as “intraceptors”. It is contemplated that the signal retentionpeptide of the present invention has the amino acid sequenceLys-Asp-Glu-Leu (KDEL; SEQ ID NO:1), which binds ER retention,receptors, preventing secretion of ligands of proteins coupled to KDEL(SEQ ID NO: 1). In further embodiments, the signal retention peptide isselected from the group consisting of KDEL (SEQ ID NO: 1), RDEL (SEQ IDNO:7), and HDEL (SEQ ID NO:8).

As used herein, the intraceptors of the present invention is preferablyproduced by recombinant DNA techniques. For example, a nucleic acidmolecule encoding an intraceptor is cloned into an expression vector,the expression vector is introduced into a host cell, and theintraceptor is expressed in the host cell. The intraceptor can then beisolated from the cells by an appropriate purification scheme usingstandard polypeptide purification techniques. For the purposes of theinvention, the term “recombinant polynucleotide” refers to apolynucleotide that has been altered, rearranged, or modified by geneticengineering. Examples include any cloned polynucleotide, andpolynucleotides that are linked or joined to heterologous sequences. Theterm “recombinant” does not refer to alterations to polynucleotides thatresult from naturally occurring events, such as spontaneous mutations.Alternative to recombinant expression, the intraceptor of the presentinvention can be synthesized chemically using standard peptide synthesistechniques. Moreover, native polypeptides for the intraceptor of thepresent invention, e.g., Flt and/or domains 2 and 3 or domains 2-4, canbe isolated from cells (e.g., human cells), for example using ananti-Flt, anti-Flt23, or anti-Flt24 polypeptide antibody.

As used herein, the term “nucleotide” and “polynucleotide” refer to RNAor DNA that is linear or branched, single or double stranded, or ahybrid thereof. The term also encompasses RNA/DNA hybrids. These termsalso encompass untranslated sequence located at both the 3′ and 5′ endsof the coding region of the gene; at least about 1000 nucleotides ofsequence upstream from the 5′ end of the coding region and at leastabout 200 nucleotides of sequence downstream item the 3′ end of thecoding region, of the gene. Less common bases, such as inosine,5-methykytosine, 6-methyladenine, hypoxanthine, and others can also beused for antisense, dsRNA, and ribozyme pairing. For example,polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression. Other modifications, such asmodification to the phosphodiester backbone, or the 2′-hydroxy in theribose sugar group of the RNA can also be made. The antisensepolynucleotides and ribozymes can consist entirely of ribonucleotides,or can contain mixed ribonucleotides and deoxyribonucleotides. Thepolynucleotides of the invention may be produced by any means, includinggenomic preparations, cDNA preparations, in vitro synthesis, RT-PCR, andin vitro or in vivo transcription.

An “isolated” nucleic acid or polynucleotide molecule is one that issubstantially separated from other nucleic acid molecules, which arepresent in the natural source of the nucleic acid (i.e., sequencesencoding other polypeptides). Preferably, an “isolated” nucleic acid isfree of some of the sequences. Which naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) inits naturally occurring replicon. For example, a cloned nucleic acid isconsidered isolated. In various embodiments, the isolated intraceptornucleic acid molecule can contain, less than about 5 kb, 4 kb, 3 kb, 2Kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived (e.g., a human or rat cell). A nucleic acidis also considered isolated if it has been altered by humanintervention, or placed in a locus or location that is not its naturalsite. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be free from some of the other cellular material withwhich it is naturally associated, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized.

Specifically excluded from the definition of “isolated nucleic acids”are: naturally-ocurring chromosomes (such as chromosome spreads),artificial chromosome libraries, genomic libraries, and cDNA librariesthat exist either as an in vitro nucleic acid preparation or as atransfected/transformed host cell preparation, wherein the host cellsare either an in vitro heterogeneous preparation or plated as aheterogeneous population of single colonies. Also specifically excludedare the above libraries wherein a specified nucleic acid mates up lessthan 5% of the number of nucleic acid inserts in the vector molecules.Further specifically excluded are whole cell genomic DNA or whole cellRNA preparations (including whole cell preparations that aremechanically sheared or enzymatically digested). Even fartherspecifically excluded are the whole cell preparations found as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis wherein the nucleic acid of the invention has notfurther been separated from the heterologous nucleic acids in theelectrophoresis medium (e.g., further separating by excising a singlehand from a heterogeneous band, population in an agarose gel or nylonblot).

A nucleic acid molecule of the present invention, or a portion thereof,can be isolated using standard molecular biology techniques and thesequence information provided herein, for example, a cDNA for domains 2and 3 or domains 2, 3, and 4 of Flt can be isolated from a cDNA libraryusing all or portion of one of the sequences encoding Flt23 (nucleotides1-633 of SEQ ID NO:3) or Flt24 (nucteotides 1-915 of SEQ ID NO:5).Moreover, a nucleic acid molecule encompassing all or a portion of Flt23(nucleotides 1-633 of SEQ ID NO:3), or Flt24 (nucleotides 1-915 of SEQID NO:5) can be isolated by the polymerase chain reaction (PCR) usingoligonucleotide primers designed based upon the sequences. For example,mRNA can be isolated from a cell, and synthetic oligonucleotide primersfor PCR amplification can be designed based upon one of the nucleotidesequences of Flt23 (nucleotides 1-633 of SEQ ID NO:3), or Flt24(nucleotides 1-915 of SEQ ID NO: 5). A nucleic acid molecule of thepresent invention can be amplified using cDNA or, alternatively, genomicDNA, as a template and appropriate oligonucleotide primers according tostandard PCR amplification techniques. The nucleic acid molecule soamplified can be cloned into an appropriate vector and characterised byDNA sequence analysis. Furthermore, oligonucleotides corresponding tothese nucleotide sequences can be prepared by standard synthetictechniques, e.g., using an automated DNA synthesizer.

The present invention provides an isolated nucleic acid, wherein thenucleic acid comprises a polynucleotide selected from the groupconsisting of: a) a polynucleotide as defined in SEQ ID NO:3; b) apolynucleotide as defined in SEQ ID NO:5; c) a polynucleotide encoding apolypeptide as defined in SEQ ID NO:4; d) a polynucleotide encoding apolypeptide as defined in SEQ ID NO:6; and e) a polynucleotidecomplementary to a full-length polynucleotide of any one of a) throughd) above. In one embodiment, an isolated nucleic acid molecule of thepresent invention comprises one of the polynucleotide sequences shown inSEQ ID NO:3 or SEQ ID NO:5. In another preferred embodiment, an isolatednucleic acid molecule of the present invention comprises apolynucleotide sequence encoding a polypeptide as shown in SEQ ID NO:4or SEQ ID NO:6. In yet another embodiment, the invention provides anisolated nucleic acid comprising a polynucleotide encoding a polypeptidehaving at least 80% sequence identity with a polypeptide as shown in SEQID NO:4 or SEQ ID NO:6, and wherein the nucleic acid may interacts withVEGF and/or a VEGFR. and can decrease the activity of VEGF and/or theVEGFR in a further embodiment, the invention provides an isolatednucleic acid, wherein the nucleic acid comprises a polynucleotide thathybridizes under highly stringent conditions to a second nucleic acidselected from the group consisting of: a) a polynucleotide as defined inSEQ ID NO: 3 or SEQ ID NO:5; and b) a polynucleotide encoding apolypeptide as defined in SEQ ID NO:4 or SEQ ID NO:6, wherein saidnucleic acid encodes a polypeptide that interacts with VEGF and/orVEGFR-2, and wherein the stringent conditions comprise hybridization ina 6×SSC solution at 65° C. In one embodiment of the present invention,the isolated nucleic acids encode a polypeptide that is capable ofinteracting with VEGF and/or a VEGFR.

Moreover, the nucleic acid molecule of the present invention cancomprise a portion, of one of the sequences of Flt23 (nucleotides 1-633of SEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ ID NO:5), forexample, a fragment that can be used as a probe or primer or a fragmentencoding a biologically active portion of the intraceptor of the presentinvention. The nucleotide sequences determined from the cloning of theVEGF, and/or VEGFR genes from human cells allow for the generation ofprobes and primers designed for use in identifying and cloning theintraceptor homologs from other cell types and organisms.

As used herein, the term “biologically active portion of” theintraceptor polypeptide is intended to include a portion, e.g., adomain/motif, of a Flt23 (amino acids 1-211 of SEQ ID NO:4), or Flt24(amino acids 1-305 of SEQ ID NO:6) polypeptide that participates in theinteraction with of the intraceptor with VEGF and/or a VEGFR.Biologically active portions of a Flt23 (amino acids 1-211 of SEQ IDNO:4), or Flt24 (amino acids 1-305 of SEQ ID NO:6) polypeptide includepeptides comprising amino acid sequences derived from the amino acidsequence of a Flt23 (amino acids 1-211 of SEQ ID NO:4), or Flt24 (aminoacids 1-305 of SEQ ID NO:6) polypeptide, or the amino acid sequence of apolypeptide identical to a Flt23 (amino acids 1-211 of SEQ ID NO:4), orFlt24 (ammo acids 1-305 of SEQ ID NO:6) polypeptide which include feweramino acids than a full length of those polypeptides, or the full lengthpolypeptide which is identical to a Flt23 (amino acids 1-211 of SEQ IDNO:4), or Flt24 (ammo acids 1-305 of SEQ ID NO: 6) polypeptide, andexhibit at least one activity of a Flt23 (amino acids 1-211 of SEQ IDNO:4), or Flt24 (amino acids 1-305 of SEQ ID NO:6) polypeptide.Typically, biologically active portions (e.g., peptides which, ate, forexample, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90,or more amino acids in length) comprise a domain or motif with at leastone activity of a Flt polypeptide. As used herein, the term “activity”is intended to Include, but is not limited to, the interaction with aVEGFR or VEGF to modulate VEGF signaling. As also used herein, the term“interaction with a VEGFR or VEGF” is intended to include, but is notlimited to, the binding of the intraceptors of the present Invention toa VEGFR. or to VEGF to sequester the protein, or decrease the expressionor secretion of the VEGF and/or VEGFR.

The present invention also provides intraceptors, e.g., Flt23K (SEQ IDNO:4) or Flt24R (SEQ ID NO:6), comprising chimeric or fusionpolypeptides. As used herein, a “chimeric polypeptide” or “fusionpolypeptide” comprises at least a portion of an extracellular receptoroperatively linked to another substantially different peptide. One ofthe intraceptors of the present invention, namely, Flt23K polypeptide(SEQ ID NO:4), refers to a polypeptide having an amino acid sequencecorresponding to domains 2 and 3 of VEGFR (Flt) polypeptide (amino acids1-211 of SEQ ID NO:4), and a signal retention peptide having an aminoacid sequence Lys-Asp-Glu-Leu (KDEL; SEQ ID NO:1). Another intraceptorof the present invention, namely, Flt24K polypeptide (SEQ ID NO:6),refers to a polypeptide having an amino acid sequence corresponding todomains 2, 3 and 4 of VEGFR-1 (Flt) polypeptide (amino acids 1-305 ofSEQ ID NO:6), and a signal retention peptide KDEL (SEQ ID NO:1. As usedherein, the term “operatively linked” is intended to indicate mat theFlt23 (amino acids 1-211 of SEQ ID NO:4) or Flt24 (amino acids 1-30.5 ofSEQ ID NO:6) polypeptide and the signal retention peptide, KDEL (SEQ IDNO: 1), are fused to each other so that both sequences fulfill theproposed function attributed to the sequence used. The signal retentionpeptide can be fused to the N-terminus or C-terminus of the VEGFR-1polypeptide. For example, in one embodiment, the intraceptors of thepresent invention are fusion polypeptides, Flt23-KDEL (Flt23K; SEQ IDNO:4) and Flt24-KDEL (Flt24K; SEQ ID NO:6), in which the Flt23 (aminoacids 1-213 of SEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6)sequences are fused with KDEL (SEQ ID NO: 1) at the C-terminus.

The invention further encompasses an intraceptor comprising apolypeptide encoded by a nucleotide sequence encoding at least a portionof an extracellular receptor, operatively linked to a signal retention,peptide. As used herein, “extracellular receptor” refers to a receptorthat has an extracellular domain. Such receptors can also havetransmembrane and intracellular domains, or can be otherwise tethered tothe cell. Non-limiting examples of extracellular receptors includereceptor kinases such as EGF or TGF-beta receptors, G-coupled proteinreceptors such as the V2vasopressin receptor, and ligand-gated ionchannels such as the nicotinic cholinergic receptor.

Preferably, the intraceptors of the present invention comprising achimeric or fusion polypeptide are produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques, for example by employing blunt-ended, orstagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirable joiningand enzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (See,for example, Current Protocols in Molecular Biology, Eds. Ausubel et al.John Wiley & Sons: 1992), Moreover, many expression vectors arecommercially available that already encode a fusion moiety.

In addition to fragments and fusion polypeptides of the intraceptorsdescribed herein, the present invention includes homologs and analogs ofnaturally occurring Flt23 (amino acids 1-211 of SEQ ID NO:4) or Flt24(amino acids 1-305 of SEQ ID NO:6) polypeptides and Flt3 (nucleotides1-633 of SEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ ID NO: 5)encoding nucleic acids in the same or other organisms. “Homologs” aredefined herein as two nucleic acids or polypeptides that have similar or“identical,” nucleotide or amino acid sequences, respectively. Homologsinclude allelic variants, orthologs, paralogs, agonists, and antagonistsof Flt23 (amino acids 1-211 of SEQ ID NO:4) and Flt24 (amino acids 1-305of SEQ ID NO:6) polypeptides as defined hereafter. The term “homolog”further encompasses nucleic acid molecules that differ from one of thenucleotide sequence of Flt (SEQ ID NO: 12), Flt23 (nucleotides 1-633 ofSEQ ID NO:3), or Flt24 (nucleotides 1-915 of SEQ ID NO: 5) (and portionsthereof) due to degeneracy of the genetic code and thus encode the sameFlt23, Flt24, Flt23K or Flt24K polypeptide as that encoded by thenucleotide sequences shown in SEQ ID NO:3 or SEQ ID NO:5, or portionsthereof. As used herein, a “naturally occurring” Flt23 (amino acids1-211 of SEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO: 6)polypeptide refers to a Flt23 or Flt24 amino acid sequence that occursin nature. Preferably, a naturally occurring Flt23 or Flt24 polypeptidecomprises an amino acid sequence as defined in SEQ ID NO:4 or SEQ IDNO:6 minus the KDEL (SEQ ID NO: 1) sequence at the C-terminal.

Art agonist of the intraceptors of the present invention can retainsubstantially the same, or a subset, of the biological activities of theintraceptors of the present invention. An antagonist of the intraceptorsof the present invention polypeptide can inhibit one or more of theactivities of the intraceptor.

Nucleic acid molecules corresponding to natural allelic variants andanalogs, orthologs, and paralogs of a Flt (SEQ ID NO: 12), Flt23(nucleotides 1-633 of SEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ IDNO: 5) cDNA can be isolated based on their identity to the Flt, Flt23 orFlt24 nucleic acids described herein using Flt, Flt23 or Flt24 cDNAs, ora portion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions. In analternative embodiment, homologs of the Flt, Flt23 or Flt24 polypeptidecan be identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the Flt, Flt23, or Flt24, for Flt, Flt23 or Flt24agonist or antagonist activity. In one embodiment, a variegated libraryof Flt, Flt23 or Flt24 variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of Flt, Flt23, or Flt24 variants canbe produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential Flt, Flt23 or Flt24 sequences is expressible asindividual polypeptides, or alternatively, as a set of larger fusionpolypeptides (e.g., for phage display) containing the set of Flt, Flt23or Flt24 sequences therein. There are a variety of methods that can beused to produce libraries of potential Flt, Flt23 or Flt24 homologs froma degenerate oligonucleotide sequence. Chemical synthesis of adegenerate gene sequence can be performed in an automatic DNAsynthesizer, and the synthetic gene is then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential Flt, Flt23 or Flt24 sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (See, e.g., Narang,1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem 53:323;Itakura et al., 1984, Science 198: 1056; Ike et al., 1983, Nucleic AcidRes. 11:477).

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of Flt, Flt23 or Flt24homologs. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing fee combinatorial genes under conditions in whichdetention of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique that enhances the frequency of functionalmutants in the libraries, can be used in combination with the screeningassays to identify Flt, Flt23 or Flt24 homelogs (Arkin & Yourvan, 1992,PNAS 89:7811-7315; Delgrave et al., 1993, Polypeptide Engineering6(3):327-331),

As stated above, the present invention includes intraceptors, e.g,Flt23K (SEQ ID NO:4) and Flt24 (SEQ ID NO:6), which comprise Flt23 andFlt24 polypeptides operatively linked to a signal retention peptide, andhomologs thereof. To determine the percent sequence identity of twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of onepolypeptide for optimal alignment with the other polypeptide or nucleicacid). The amino acid residues at corresponding amino acid positions arethen compared. When a position in one sequence is occupied by the sameamino acid residue as the corresponding position in the other sequence,then the molecules are identical at that position. The same type ofcomparison can be made between two nucleic acid sequences.

The percent sequence identity between the two sequences is a function ofthe number of identical positions shared by the sequences (i.e., percentsequence identity=numbers of identical positions/total numbers ofpositions×100). Preferably, the isolated amino acid homologs included inthe present invention are at least about 50-60%, preferably at leastabout 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90%. or 90-95%, and most preferably at least about 96%, 97%, 98%,99%, or more identical to an entire amino acid sequence of Flt23 (aminoacids 1-211 of SEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6). In yet another embodiment, the isolated amino acid homologs includedin the present invention are at least about 50-60%, preferably at leastabout 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%,99%, or more identical to an entire amino acid sequence encoded by anucleic acid sequence of Flt23 (nucleotides 1-633 of SEQ ID NO:3) orFlt24 (nucleotides 1-915 of SEQ ID NO:5). In other embodiments, theamino acid homologs have sequence identity over at least 15 contiguousamino acid residues, more preferably at least 25 contiguous amino acidresidues, and most preferably at least 35 contiguous amino acid residuesof Flt23 (amino acids 1-211 of SEQ ID NO:4) or Flt24 (amino acids 1-305of SEQ ID NO:6). In another embodiment, the homologs of the presentinvention are preferably at least about 60-70%, and more preferably atleast about: 80-85%, 85-90%, or 90-95%, and most preferably at leastabout 96%, 97%, 98%, 99%, or more identical to Flt23 (amino acids 1-211of SEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6).

In another preferred embodiment, an isolated nucleic acid homolog of theinvention comprises a nucleotide sequence which is at least about60-70%, more preferably at least about 70-75%, 75-89%, 80-85%, 85-99%,or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%,99%, or more identical to a nucleotide sequence of Flt23 (nucleotides1-633 of SEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ ID NO: 5), orto a portion comprising at least 60 consecutive nucleotides thereof. Inone embodiment, the Flt23 or Flt24 homolog nucleotide sequence is about80-90% identical to a nucleotide sequence of Flt23 (nucleotides 1-633 ofSEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ ID NO:5). The preferablelength of sequence comparison for nucleic acids is at least 75nucleotides, more preferably at least 100 nucleotides, and mostpreferably the entire length of the coding region for Flt23 or Flt24. Itis even more preferable that the nucleic acid homologs encode proteinshaving homology with Flt23 (amino acids 1-211 of SEQ ID NO:4) or Flt24(amino acids 1-305 of SEQ ID NO:6).

It is further preferred that the isolated nucleic acid homolog of theinvention encodes a Flt33 or Flt24, or portion thereof, that is at least80% identical to an amino acid sequence of Flt23 (amino acids 1-211 ofSEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6), and that,after coupled with a signal retention peptide, KDEL (SEQ ID NO:1), mayfunction by interacting with a VEGF and/or a VEGFR to thereby disruptthe VEGF signaling pathway.

For the purposes of the invention, the percent sequence identify betweentwo nucleic acid or polypeptide sequences is determined using the VectorNTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Sethesda,Md. 20814). A gap opening penalty of 15 and a gap extension penalty of6.66 are used for determining the percent identity of two nucleic acids.A gap opening penalty of 10 and a gap extension penalty of 0.1 are usedfor determining the percent identity of two polypeptides. All otherparameters are set at the default settings. For purposes of a multiplealignment (Clustal W algorithm), the gap opening penalty is 10, and thegap extension penalty is 0.05 with blosum62 matrix. It is to heunderstood that for the purposes of determining sequence identity whencomparing a DNA sequence to an RNA sequence, a thymidine nucleotide isequivalent to a uracil nucleotide.

In another aspect, the invention provides an intraceptor encoded by anisolated nucleic acid comprising a polynucleotide that hybridizes to thepolynucleotide of Flt23 (nucleotides 1-633 of SEQ ID NO:3) or Flt24(nucleotides 1-915 of SEQ ID NO: 5) under stringent conditions. Moreparticularly, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence of Flt23(nucleotides 1-633 of SEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ IDNO:5). In other embodiments, the nucleic acid is at least 30, 50, 100,250, or more nucleotides in length. Preferably, an isolated nucleic acidhomolog of the invention comprises a nucleotide sequence whichhybridizes under highly stringent conditions to the nucleotide sequenceof Flt23 (nucleotides 1-633 of SEQ ID NO:3) or Flt24 (nucleotides 1-915of SEQ ID NO: 5), and after coupled with a signal retention peptide, itmay be used, to interact with, a VEGF and/or VEGFR for the treatment ofangiogenesis.

As used herein with regard to hybridization for DNA to a DNA blot, theterm “stringent conditions” refers to hybridization overnight at 6° C.in 10× Denhardt's solution, 6×SSC, 0.5% SDS, and 100 μg/ml denaturedsalmon sperm DNA, Blots are washed sequentially at 65° C. for 30 minuteseach time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS, and finally0.1×SSC/0.1% SDS. As also used herein, “highly stringent conditions”refers to hybridization overnight at 65° C. in 10× Denhardt's solution,6×SSC, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. Blots arewashed sequentially at 65° C. for 30 minutes each time in 3×SSC/0.1%SDS, followed by 1×SSC/0.1% SDS, and finally 0.1×SSC/0.1% SDS. Inanother embodiment, “highly stringent conditions” refers tohybridization at 65° C. in a 6×SSC solution. Methods for nucleic acidhybridizations are described in Meinkoth and Wahl 1984, Anal. Biochem.138: 267-284; Current Protocols in Molecular Biology, Chapter 2,Asusubel et al., Greene Publishing and Wiley-Interscience, New York,1995; and Tijssen, 1993, Laboratory Techniques in Biochemistry andMolecular Biology; Hybridization with Nucleic Acid Probes, Part 1Chapter 2, Elsevier, New York, 1993. Preferably, an isolated nucleicacid molecule of tins invention that hybridizes under stringent orhighly stringent conditions to a sequence of Flt23 (nucleotides 1-633 ofSEQ ID NO:3) or Flt24 (nucleotides 1-915 of SEQ ID NO:5) corresponds toa naturally occurring nucleic acid molecule. As used herein, a“naturally occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural, polypeptide).

Using the above-described methods, and others known to those of skill isthe art, one of ordinary skill in the art can isolate homologs of Flt23or Flt24 polypeptides comprising amino acid sequences of Flt23 (aminoacids 1-211 of SEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6),respectively. One subset of these homologs is allelic variants. As usedherein, the term, “allelic variant” refers to a nucleotide sequencecontaining polymorphisms that lead to changes in the amino acidsequences of Flt23 (amino acids 1-211 of SEQ ID NO:4) or Flt24 (aminoacids 1-305 of SEQ ID NO:6) and that exist within a natural population,Such natural allelic variations can typically result in 1-5% variance ina Flt23 or Flt24 nucleic acid. Allelic variants can be identified bysequencing the nucleic acid sequence of interest in a number ofdifferent organisms, which can be readily carried out by usinghybridization probes to identify the same Flt23 or Flt24 genetic locusin those organisms. Any and all such nucleic acid variations andresulting amino acid polymorphisms or variations in a Flt23 or Flt24polypeptide that are the result of natural allelic variation and that donot alter the functional activity of a Flt23 or Flt24 polypeptide, areintended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding Flt23 or Flt24 polypeptidesfrom, the same or other species such as Flt23 or Flt24 analogs,orthologs, and paralogs, are intended to be within the scope of thepresent invention. As used herein, the term “analogs” refers to twonucleic acids that have the same or similar function, but that haveevolved separately in unrelated organisms. As used herein, the term“orthologs” refers to two nucleic acids from different species, but thathave evolved from a common ancestral gene by speciation. Normally,orthologs encode polypeptides having the same or similar functions. Asalso used herein, the term “paralogs” refers to two nucleic acids thatare related by duplication within a genome. Paralogs usually havedifferent functions, but these functions may be related (Tatusov, R. L.et al., 1997, Science 278(5338):631-637). Analogs, orthologs, andparalogs of a naturally occurring Flt23 or Flt24 polypeptide can differfrom the naturally occurring Flt23 or Flt24 polypeptide bypost-translational modifications, by amino acid sequence differences, orby both. Post-translational modifications include in vivo and in vitrochemical derivatization of polypeptides, e.g., acetylation,carboxylation, phosphorylation, or glycosylation, and such modificationsmay occur during polypeptide synthesis or processing or followingtreatment with isolated modifying enzymes. In particular, orthologs ofthe invention will generally exhibit at least 80-85%, more preferably,85-90% or 90-95%, and most preferably 95%, 96%, 97%, 98%, or even 99%identity, or 100% sequence identity, with all or part of a naturallyoccurring Flt23 or Flt24 amino acid sequence, and will exhibit afunction similar to a Flt23 or Flt24 polypeptide. Preferably, a Flt23 orFlt24 ortholog of the present invention is encoded by a nucleic acidthat may be used to interact with VEGF and/or a VEGFR to thereby disruptthe VEGF signaling pathway.

In addition to naturally-occurring variants of a Flt, Flt23 or Flt24sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into anucleotide sequence of Flt, Flt23 or Flt24, thereby leading to changesin the amino acid sequence of the encoded Flt, Flt23 orFlt24polypeptide, without altering the functional activity of the Flt,Flt23 or Flt24polypeptide. For example, nucleotide substitutions leadingto amino acid substitutions at “non-essential” amino acid residues canbe made in a sequence of Flt, FIt23 or Flt24. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequence of one of the Flt, Flt23 or Flt24 polypeptides without alteringthe activity of said Flt, FIt23 or Flt24 polypeptide, whereas an“essential” amino acid residue is required for Flt, Flt23 or FIt4activity. Other amino acid residues, however, (e.g., those that are notconserved or only semi-conserved in the domain having Flt, Flt23 orFlt24 activity) may not he essential for activity and thus are likely tobe amenable to alteration without altering Flt, Flt23 or Flt24 activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding Flt, Flt23 or Flt24 polypeptides coupled with asignal retention peptide, such as KDEL (SEQ ID NO: 1), RDEL (SEQ IDNO:7), or HDEL (SEQ ID NO:8), wherein the Flt, Flt23 or Flt24polypeptides contain changes in amino acid residues that are notessential for Flt, Flt23 or Flt24 activity. Such Flt, Flt23 orFlt24polypeptides may have different amino acid sequence, yet retain atleast one of the Flt Flt23 or Flt24 activities described herein, in oneembodiment, the isolated nucleic add molecule comprises a nucleotidesequence encoding a polypeptide, wherein the polypeptide comprises anamino acid sequence at least about 89% identical to an amino acidsequence of Flt (SEQ ID NO:13), Flt23 (amino acids 1-211 of SEQ ID NO:4)or Flt24 (amino acids 1-305 of SEQ ID NO:6). Preferably, the polypeptideencoded by the nucleic-acid molecule is at least about 80-85% identicalto one of the sequences of Flt (SEQ ID NO:13), Flt23 (amino acids 1-211of SEQ ID NO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6), morepreferably at least about 88-90% or 90-95% identical to one of thesequences of Flt (SEQ ID NO: 13), FIt23 (amino acids 1-211 of SEQ IDNO:4) or Flt24 (amino acids 1-305 of SEQ ID NO:6), and most preferablyat least about 96%, 97%, 98%, or 99% identical to one of the sequencesof Flt (SEQ ID NO:13), Flt23 (amino acids 1-211 of SEQ ID NO:4) or Flt24(amino acids 1-365 of SEQ ID NO:6).

The intraceptors of the present invention, Flt23K (SEQ ID NO:4) orFlt24K (SEQ ID NO:6), encoded by an isolated nucleic acid moleculehaving sequence identity with a polypeptide sequence of Flt, Flt23,Flt24, Flt23K, or Flt24K can be created by introducing one or morenucleotide substitutions, additions or deletions into a nucleotidesequence of Flt (SEQ ID NO: 12 or 13), Flt23 (nucleotides 1-633 of SEQID NO:3) or Flt24 (nucleotides 1-915 of SEQ ID NO:5), respectively, suchthat one or more amino acid substitutions, additions, or deletions areintroduced into the encoded polypeptide. Mutations can be introducedinto one of the sequences of Flt (SEQ ID NO: 12 or 13), Flt23(nucleotides 1-633 of SEQ ID NO:3), Flt24 (nucleotides 1-915 of SEQ IDNO:5), Flt23K (SEQ ID NO:3) or Flt24K (SEQ ID NO:5) by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine), and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a Flt (SEQ ID NO: 13),Flt23 (amino acids 1-211 of SEQ ID NO:4), Flt24 (amino acids 1-305 ofSEQ ID NO:6), Flt23K (SEQ ID NO:4) or Flt24K (SEQ ID NO:6) polypeptideis preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a Flt, Flt23, Flt24, Flt23K,or Flt24K sequence, such, as by saturation mutagenesis, and theresultant mutants can be screened for a Flt. Flt23, Flt24, Flt23K orFlt24K activity described herein to identify mutants that retain theactivity. Following mutagenesis of one of the sequences of Flt, Flt23,Flt24, Flt23K, Flt24K, the encoded polypeptide can be expressedrecombinantly and the activity of the polypeptide can be determined.

Additionally, optimized nucleic acids encoding intraceptors can becreated. Preferably, an optimised nucleic acid encodes an intraceptorcomprising a Flt23 or Flt24 polypeptide coupled with signal retentionpolypeptide feat binds to VEGF and/or a VEGFR and, in one embodimentsuppresses the expression and/or secretion of VEGF or the VEGFR. As usedherein, “optimized” refers to a nucleic acid that is geneticallyengineered, to increase its expression in a given organism. To provideoptimised intraceptor nucleic acids (e.g., Flt23K, (SEQ ID NO:3) andFlt24K, (SEQ ID NO:5), the DNA sequence of the gene can be modifiedto 1) comprise codons preferred by highly expressed genes in theorganism; 2) comprise an A+T content in nucleotide base composition tothat substantially found in the organism; 3) form an initiation sequencefor that organism; or 4) to eliminate sequences that causedestabilization, inappropriate polyadenylation, degradation andtermination of RNA, or that form secondary structure hairpins or RNAsplice sites. Increased expression of intraceptor nucleic acids (e.g.Flt23K (SEQ ID NO:3) and Flt24K: (SEQ ID NO:5)) in an organism can beachieved by utilizing the distribution frequency of codon usage in aparticular organism.

As used herein, “frequency of preferred codon usage” refers to thepreference exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. To determine the frequency ofusage of a particular codon in a gene, the number of occurrences of thatcodon in the gene is divided by the total number of occurrences of allcodons specifying the same amino acid in the gene. Similarly, thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell it is preferable that ibisanalysis be limited to genes that are highly expressed by the host cell.The percent deviation of the frequency of preferred codon usage for asynthetic gene from that employed by a host cell is calculated first bydetermining the percent deviation of the frequency of usage of a singlecodon front that of the host cell followed by obtaining the averagedeviation over all codons. As defined herein, this calculation includesunique codons (i.e., ATG and TGG). In general terms, the overall averagedeviation of the codon usage of an optimized gene from, that of a hostcell is calculated using the equation 1A=n=1 Z X_(N)−Y_(N)X_(N) times100 Z where X_(N)=frequency of usage for codon n in the host cell;Y_(N)=frequency of usage for codon n in the synthetic gene; n representsan individual codon that specifies an amino acid; and the total numberof codons is Z. The overall deviation of the frequency of codon usage.A, for all amino acids should preferably be less than, about 25%, andmore preferably less than about 10%.

Hence, an intraceptor nucleic acid, e.g. Flt23K (SEQ ID NO:3) and Flt24K(SEQ ID NO:5) can he optimized such that its distribution frequency ofcodon usage deviates, preferably, no more than 25% from that of highlyexpressed genes in that organism and, more preferably, no more thanabout 10%. In addition, consideration is given to the percentage G+Ccontent of the degenerate third base.

In addition to the intraceptor nucleic acids, Flt23K, SEQ ID NO:3 andFlt24K, SEQ ID NO:5, and their encoding polypeptides described above,the present invention encompasses these nucleic acids and polypeptidesattached to a moiety. These moieties include, but are not limited to,detection moieties, hybridization moieties, purification moieties,delivery moieties, reaction moieties, binding moieties, and the like. Atypical group of nucleic acids having moieties attached are probes andprimers. Probes and primers typically comprise a substantially isolatedoligonucleotide. As used herein, the terms “probe” and “primer” areintended to include oligonucleotides that typically comprise a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50, or 75consecutive nucleotides of a sense strand of one of the sequences of Flt(SEQ ID NO: 12 or 13). Flt23 (nucleotides 1-633 of SEQ ID NO:3), Flt24(nucleotides 1-915 of SEQ ID NO:5), Flt23K (SEQ ID NO:3), or Flt24 (SEQID NO:5); an anti-sense sequence of one of the sequences of Flt (SEQ IDNO: 12 or 13), Flt23 (nucleotides 1-633 of SEQ ID NO:3), Flt24(nucleotides 1-915 of SEQ ID NO:5), Flt23K (SEQ ID NO:3), or Flt24K (SEQID NO:5); or naturally occurring mutants thereof. Primers based on anucleotide sequence of Flt (SEQ ID NO: 12 or 13), Flt23 (nucleotides1-633 of SEQ ID NO:3), Flt24 (nucleotides 1-915 of SEQ ID NO:5), Flt23K(SEQ ID NO:3), or Flt24K (SEQ ID NO:5) can be used in PCR reactions toclone Flt (SEQ ID NO: 12 or 13), Flt23 (nucleotides 1-633 of SEQ IDNO:3), Flt24 (nucleotides 1-915 of SEQ ID NO:5), Flt23K (SEQ ID NO:3),or Flt24K (SEQ ID NO:5) homologs. Probes based on these nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or substantially identical polypeptides. In preferredembodiments, the probe further comprises a label group attached thereto,e.g. the label group can be a radioisotope, a bioluminescent compound, achemiluminescent compound, a metal chelate, a fluorescent compound, anenzyme, or an enzyme co-factor.

In particular, a useful method to ascertain the level of transcription,of the gene (an indicator of the amount of mRNA available fortranslation to the gene product) is to perform a Northern blot (Forreference, see, for example, Ausubel et al., 1988, Current Protocols inMolecular Biology, Wiley: New York). The information from a Northernblot at least partially demonstrates the degree of transcription of thetransformed gene. Total cellular RNA cart be prepared from cells,tissues, or organs by several methods, all well-known in the art, suchas that described, in Bormann E. R. et al., 1992, Mol. Microbiol6:317-326. To assess tire presence or relative quantity of polypeptidetranslated from this mRNA, standard techniques, such as a Western blot,may be employed. These techniques are well known to one of ordinaryskill in the art. (See, for example, Ausubel et al. 1988, CurrentProtocols in Molecular Biology, Wiley: New York).

The invention further provides an isolated recombinant expression vectorcomprising the intraceptor nucleic acids as described above, whereinexpression of the intraceptor nucleic acid in a host cell results inmodulation of VEGF and/or VEGFR activity as compared to a wild typevariety of the host cell. As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid.” whichrefers to a circular double stranded DNA loop into which additional DMAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host, cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors” In general, expression vectors of utility. In recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as feeplasmid is the most commonly uses form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication detective retroviruses, adenoviruses,and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention, comprise thenucleic acid for the intraceptors of the present invention in a formsuitable for expression of the nucleic acid, in a host cell, which meansthat the recombinant expression, vectors include one or more regulatorysequences, selected on the basis of the host cells to be used forexpression, which is operatively linked to the nucleic acid sequence tobe expressed. As used herein with respect to a recombinant expressionvector, “operatively linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequencers) in a mannerwhich allows for expression, of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers, and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruberand Crosby, in: Methods in Plant Molecular Biology and Biotechnology,eds. Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Fla.,including the references therein. Regulatory sequences include thosethat direct constitutive expression of a nucleotide sequence in manytypes of host cells and those that direct expression of the nucleotidesequence only in certain host cells or under certain conditions. It willbe appreciated by these skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce polypeptides or peptides, including fusionpolypeptides or peptides, encoded by nucleic acids as described herein(e.g., Flt23, Flt24, Flt23K, Flt24K polypeptides, mutant forms orvariants thereof).

The recombinant expression, vectors of the invention can be designed forexpression of intraceptor polypeptides in prokaryotic or eukaryoticcells. For example, intraceptor Flt23K (SEQ ID NO: 3) or Flt24K (SEQ IDNO: 5) genes can be expressed in bacterial cells such as C. glutamicum,insect cells (using baculovirus expression vectors), yeast and otherfungal cells (See Romanos, M. A. et al., 1992, Foreign gene expressionin yeast: a review. Yeast 8:423-488; van den Hondel, C. A. M. J. J. etal., 1991, Heterologous gene expression in filamentous fungi, in: MoreGene Manipulations in Fungi, J. W. Bonnet & L. L. Lasure, eds., p.396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J. &Punt, P. J., 1991, Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae(Falciatore et al. 1999, Marine Biotechnology 1(3):239-251), ciliates ofthe types: Holotrichia, Peritrichta, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in PCT Application No. WO 98/01572, andmulticellular plant cells (See Schmidt, R, and Willmitzer, L., 1988,High efficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon explants. Plant Cell Rep.583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton,Fla., chapter 6/7, S.71-119 (1-993); F. F. White, B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilizations eds, Kung und R. Wu, 128-43, Academic Press; 1993;Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol, 42:205-225and references cited therein), or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press: San Diego, Calif. (1990). Alternatively,the recombinant expression, vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of polypeptides in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion polypeptides. Fusion vectorsadd a number of amino acids to a polypeptide encoded therein, usually tothe amino terminus of the recombinant polypeptide but also to theC-terminus or fused within suitable regions in the polypeptides. Suchfusion, vectors typically serve three purposes: 1) to increaseexpression of a recombinant polypeptide; 2) to increase the solubilityof a recombinant polypeptide; and 3) to aid in the purification of arecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide to enable separation of the recombinant polypeptide from thefusion moiety subsequent to purification of the fusion polypeptide. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin, and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S., 1988, Gene 67:31-40), pMAL, (NewEngland Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway,N.J.) which, fuse glutathione S-tansferase (GST), maltose E bindingpolypeptide, or polypeptide A, respectively, to the target recombinantpolypeptide. In one embodiment, the sequence of the intraceptor of thepresent invention is cloned into a pCMV expression vector to create avector encoding the intraceptor fusion polypeptides, Flt23K and Flt24K.The fusion polypeptide can be purified by affinity chromatography.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. 1988, Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybrid,trp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident.lamda. prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression is toexpress the polypeptide in a host bacteria with an impaired capacity toproteolytically cleave the recombinant polypeptide (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilisedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

In another embodiment, the expression vector expressing the intraceptorsof the present invention can be a yeast expression vector. Examples ofvectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari,et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982,Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.). Vector and methodsfor the construction of vectors appropriate for use in other fungi, suchas the filamentous fungi, include those detailed in: van den Hondel, C.A. M. J. J. & Punt, P. J., 1991, “Gene transfer systems and vectordevelopment for filamentous fungi,” in; Applied Molecular Genetics ofFungi, J. F. Peberdy, et al., eds, p. 1-28, Cambridge University Press:Cambridge.

Alternatively, the intraceptor polypeptides of the invention can beexpressed in insect cells using baculoviras expression vectors.Baculovirus vectors available for expression, of polypeptides incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., 1983, Mol. Cell Biol 3:2156-2165) and the pVL series (Lucklowand Summers, 1989, Virology 170:31-39).

In yet another embodiment, the intraceptors of the present invention areexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987,Nature 329:840) and pMT2PC (Kaufman et. al., 1987, EMBO J. 6:187-195),When used in mammalian, cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirus,and Simian Virus 40. For other suitable expression systems for bothprokuryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook,J., Fritsh, E. F., & Maniatis, T. Molecular Cloning: A LaboratoryManual, latest ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987, Genes Dev. 1068-277), lymphoid-specific promoter (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733), andimmunoglobulins (Baneiji et al., 1983, Cell 33:729-740; Queen, andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, PNAS 36:5473-5477),pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Grass, 1990, Science 249:374-379)and the fetopolypeptide promoter (Campes & Tilghman, 1989, Genes Dev.3:537-546).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrags, such as G418, hygromycin, and methotrexate. Nucleic acidmolecules encoding a selectable marker can be introduced into a hostcell on the same vector as that encoding fire intraceptor of the presentinvention or can be introduced on a separate vector. Cells stablytransfected, with the introduced nucleic acid molecule can be identifiedby, for example, antibiotic selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

According to the present invention, the introduced intraceptorpolypeptides, may be maintained in the host cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto a host cell chromosome. Alternatively, the introduced intraceptorpolypeptides may be present on an extra-chromosomal non-replicatingvector and may be transiently expressed or transiently active.

Whether present in an extra-chromosomal non-replicating vector or avector that is integrated into a chromosome, the intraceptorpolynucleotides preferably reside in a mammalian expression cassette. Amammalian expression cassette preferably contains regulatory sequencescapable of driving gene expression in mammalian cells that areoperatively linked so that each sequence can fulfill its function, forexample, termination of transcription by polyadenylation signals.

Gene expression should be operatively linked to an appropriate promoterconferring gene expression in a timely, cell specific, or tissuespecific manner. Promoters useful in the expression cassettes of theinvention include any promoter that is capable of initiatingtranscription, in a host cell. The promoter may be constitutive,inducible, developmental stage-preferred, cell type-preferred,tissue-preferred, or organ-preferred.

The nucleic acid molecules, polypeptides, polypeptide homologs, fusionpolypeptides, primers, vectors, and host cells described herein can beused in one or more of the following methods: evolutionary studies;determination of intraceptor regions required for function; modulationof intraceptor activity; and modulation of VEGF and/or VEGFR activity.

The invention further provides a recombinant expression vectorcomprising an DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a Flt, Flt23 or Flt24 mRNA. Regulatory sequencesoperatively linked to a nucleic acid molecule cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types. For instance, viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue specific, or cell type specific expressionof antisense RNA. The antisense expression vector can be in the form ofa recombinant plasmid, phagemid, or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genes,see Weintraub, H. et al., 1986, Antisense RNA as a molecular tool forgenetic analysis, Reviews--Trends in Genetics, Vol. 1 (1), and Mol etal., 1990, FEBS Letters 268:427-430.

Another aspect of the invention, pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell, but they also apply to the progeny of potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or enkaryotic cell. Forexample, the intraceptor polypeptide can be expressed in bacterial cellssuch as C. glutamicum, insect cells, fungal cells, or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells), algae,dilates, plant cells, fungi, or other microorganisms like C. glutamicum.Other suitable host cells are known to those skilled in the art.

A host, ceil of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an intraceptorpolypeptide. Accordingly, the invention further provides methods forproducing intraceptor polypeptides, e.g., Flt23K or Flt24K, using thehost cells of the invention. In one embodiment, the method comprisesculturing the host cell of invention (into which a recombinantexpression vector encoding the intraceptor polypeptide has beenintroduced, or into which genome has been introduced, a gene encoding awild-type or altered, intraceptor polypeptide) in a suitable mediumuntil the intraceptor polypeptide is produced. In another embodiment,the method further comprises isolating intraceptor polypeptides, e.g.,Flt23K, or Flt24K, from the medium or the host cell,

The intraceptor nucleic acid molecules of the present invention, arealso useful for evolutionary and polypeptide structural studies. Bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar polypeptides from other organisms,the evolutionary relatedness of the organisms can be assessed.Similarly, such a comparison permits an assessment of which regions ofthe sequence are conserved and which are not, which may aid indetermining those regions of the polypeptide that are essential for thefunctioning of the polypeptide. This type of determination is of valuefor polypeptide engineering studies and may give an indication of whatthe polypeptide can tolerate in terms of mutagenesis without losingfunction.

The present invention also provides a pharmaceutical compositioncomprising the intraceptors of the invention and a pharmaceuticallyacceptable carrier. The pharmaceutical compositions of the presentinvention are used for treating an angiogenesis-related condition bycontacting and/or administering the pharmaceutical composition to cellsor individuals that have such an angiogenesis-related condition. As usedherein, the term “treating” includes to preventing the condition, and/orameliorating the symptoms of the condition. In certain embodiment, thepharmaceutical composition decreases the expression or secretion of VEGFand/or a VEGFR in a cell that is involved in the angiogenesis-relatedcondition, wherein the intraceptor of the pharmaceutical compositioninteracts with VEGF and/or a VEGFR, preferably, VEGFR-2, resulting indisruption of intracellular VEGF pathways and/or signalings. Themechanisms of the intraceptor action can include, but are not limitedto, a) disruption of a VEGF autocrine loop, b) induction of the unfoldedprotein response (UPR) in cells which produce VEGF, resulting inselective ER stress, leading to apoptosis of vascular endothelial cells;and c) the formation of heterodimers with VEGFR-2 leading to itssequestration, thus, suppressing physiologic Flt/VEGFR-2 heterodimerformation. The pharmaceutical composition, of the present inventionprovides high specificity and affinity for the target molecule due tothe use of a receptor as the therapeutic substrate.

As used herein, the phrases “pharmaceutically or pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or a human, as appropriate. Veterinary usesare equally included within the invention and “pharmaceuticallyacceptable” formulations include formulations for both clinical and/orveterinary use. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings,antibacterial, and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances, is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. For human administration, preparations should meetsterility, pyrogenicity, and general safety and purity standards asrequired by FDA Office of Biologies standards. Supplementary activeingredients can also be incorporated into the compositions.

As used herein, the term “augiogenesis-related condition” includes, butis not limited to inflammation, stroke, hemangioma, solid tumors,leukemias, lymphomas, myelomas, metastasis, telangiectasia, psoriasisscleroderma, pyogenic granuloma, myocardial angiogenesis, plaqueneovascularization, coronary collaterals, ischemic limb angiogenesis,corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, wound healing, peptic ulcer, fractures, keloids,vasculogenesis, hematopoiesis, ovulation, menstruation, placentation,polycystic ovary syndrome, dysfunctional uterine bleeding, endometrialhyperplasia and carcinoma, endometriosis, failed implantation andsubnormal foetal growth, myometrial fibroids (uterine leiomyomas) andadenomyosis, ovarian hyperstimulation syndrome, ovarian carcinoma,melanoma, venous ulcers, acne, rosacea, warts, eczema,neurofibromatosis, tuberous sclerosis, and chronic inflammatory disease.In certain, embodiments, the angiogenesis-related condition is selectedfrom the group consisting of melanoma, diabetic retinopathy, and maculardegeneration.

As used herein, the term “contacting” or “administering” refers tovarious means of introducing the pharmaceutical composition into a cell,or into a patient. These means are well known, in the art and mayinclude, for example, injection; tablets, pills, capsules, or othersolids for oral administration; nasal solutions or sprays; aerosols,inhalants; topical formulations; liposomal forms; and the like. As usedherein, the term “effective amount” refers to an amount that will resultin the desired result and may readily he determined by one of ordinaryskill in the art.

The pharmaceutical, composition comprising intraceptor polypeptides,nucleic acids, and antibodies of the present invention may be formulatedfor parenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous, intrastromal, transdermal, orother such routes. The preparation of an aqueous composition thatcontains such a protein or antibody as an active ingredient will beknown to those of skill in the art in light of the present disclosure.Typically, such, compositions can be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for using toprepare solutions or suspensions upon the addition, of a liquid, priorto injection can also be prepared; and the preparations can also beemulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such, as bacteria and fungi.

The intraceptor compositions of the present invention can be formulatedinto a sterile aqueous composition in a neutral or salt form. Solutionsas free base or pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein), and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, trifluoroacetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine, and the like.

Suitable carriers include solvents and dispersion media containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. In many cases, it will be preferable toinclude isotonic agents, fox example, sugars, or sodium chloride. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants.

Under ordinary conditions of storage and use, all such, preparationsshould contain a preservative to prevent the growth, of microorganisms.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolongedabsorption of the injectable compositions can be brought about by theuse in fee compositions of agents delaying absorption, for example,aluminum monoslearate, and gelatin.

Prior to or upon formulation, the intraceptor compositions of thepresent invention should be extensively dialyzed to remove undesiredsmall molecular weight molecules, and/or lyophilized for more readyformulation into a desired vehicle, where appropriate. Sterileinjectable solutions are prepared by incorporating the active agents inthe required amount in fee appropriate solvent with various of the otheringredients enumerated above, as desired, followed by filtersterilisation. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle thatcontains the basic dispersion medium and fee required other ingredientsfrom those enumerated above.

In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques that yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Suitable, pharmaceutical compositions in accordance with the inventionwill generally include an amount of the polypeptide or nucleic acidadmixed with an acceptable pharmaceutical diluent or excipient, such asa sterile aqueous solution, to give a range of final concentrations,depending on the intended use. The techniques of preparingpharmaceutical compositions are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. It should beappreciated that for human administration, preparations should meetsterility, pyrogenicity, and general safety and parity standards asrequired by FDA Office of Biological Standards.

In one embodiment, the present invention also encompasses a method forcontrolling and/or modulating the activity, expression or secretion ofVEGF and/or VEGFR, preferably VEGFR-2. In another embodiment, thepresent invention provides a method for treating an angiogenesis-relatedcondition using the intraceptors of the present invention, and/or apharmaceutical composition comprising the same. Preferably, with respectto these methods of the present invention, an effective amount of theintraceptors and/or the pharmaceutical composition comprising theintraceptors of the present invention is administered to a cell or apatient that is involved in an angiogenesis-related pathology, providinga disruption of VEGF pathways for angiogenesis. In one embodiment, theintraceptors of the present invention comprise a polypeptide encoded bya polynucleotide selected from the group consisting of a polynucleotideas defined in SEQ ID NO:3 or SEQ ID NO:3, a polynucleotide encoding apolypeptide as defined in SEQ ID NO:4 or SEQ ID NO:6, and apolynucleotide complementary to a full-length polynucleotide thereof.

The present invention provides that the intraceptors can disrupt VEGFautocrine loop and in certain embodiments, can decrease cornealangiogenesis. In one embodiment, the present invention provides that theintraceptors are more effective at inhibiting corneal neovascularizationthan extracellular VEGF blockage such as that caused by an anti-VEGFantibody; and the intraceptors of the present invention regress cornealneovascularization. In a further embodiment, the present inventionprovides that one of the intraceptors, Flt24K (SEQ ID NO:6) suppressesVEGFR-2 expression and angiogenic events in human dermal microvascularendothelial cells (HMECs) by heterodimerization with VEGFR-2, thusleading to its sequestration. The present invention also provides thatthe intraceptor, Flt24K, can heterodimerize with VEGFR-2 after binding,with VEGF, as the full FLT receptor is known to do; such functiongreatly enhances its ability to disrupt intracellular autocrine loops,by entrapping both VEGF and its principal angiogenic receptor, VEGFR-2within the endoplasmic reticulum. VEGFR-2 is the principal receptorresponsible for VEGF-induced vascular endothelial cell proliferation andmigration due to its strong tyrosine kinase activity.

In further preferred embodiments, the Flt23K and Flt24K intraceptorsubstantially inhibit and regress corneal neovascularization in vivoalter murine corneal injury.

Alternatively, the present invention also includes additional domains ofFLT (SEQ ID NO: 13) other than domains 2, 3, and 4 complexed with KDEL(SEQ ID NO:1), or with different endoplasmic reticulum retentionsequence (e.g., HDEL (SEQ ID NO:8), RDEL (SEQ ID NO:7)). Furthermore,the present invention provides a development of an adeno- or lentivirusdirecting intraceptor production for sustained expression. Thespecificity of the intraceptors of the present invention is validated bydeveloping alternate intraceptors to ensure that intraceptors as a groupdo not suppress neovascularization.

The present invention also provides different approaches to disrupt VEGFpathways intracellularly. For instance, siRNAs (short interfering RNAs)are developed, which successfully downregulate VEGF expression andinhibit and regress injury-induced corneal NV, and/or inhibit VEGFR-2expression.

The present invention provides that accumulation of sequesteredintraceptor-VEGF complexes in the endoplasmic reticulum may lead toendoplasmic reticulum, overload, triggering the unfolded protein,response (UPR), which by itself or in combination with downregulation ofVEGF could cause apoptosis of endothelial cells. The presence ofunfolded proteins in endoplasmic reticulum (ER) leads to a stressresponse, initiated by ATF6α and IRE-1 (Wang et al., 1998, EMBO J.17:5708-17; Yoshida et al., 2001, Cell 107; 881-91; Tirasophon et al.,1998, Genes Dev. 12:1812-24), ATF6α undergoes proteolysis to p5OATF6α, atranscription factor which induces X-box binding protein 1 (XBP-1) andCHOP, which is associated with apoptosis (Fornace et al., 1988, ProcNatl Acad Set USA 85:8800-4). IRE-1, an ER transmembraneendoribonuclease and kinase, oligomerizes in the presence of ER stress,and splices XBP-1 to its active form which induces several targetfactors of endoplasmic reticulum associated degradation (ERAD) pathways(Wang et. al., 1998, EMBO J. 17; 5708-17; Tirasophon et al., 1998, GenesDev. 12:1812-24) including BiP, an ER chaperone protein, and possible“master switch” In the UPR. The effect of KDEL (SEQ ID NO:1)-mediatedprotein retention on these molecular responses is unknown, although, ithas been reported that the KDEL receptor is involved in the ER stressresponse (Yamamoto et al., 2003, J Biol Chem, 278:34525-32).

The present invention provides that UPR activation, by intraceptorsleads to clearance of retained target proteins VEGF and VEGFR-2 by theubiquitin-proteasome or lysosome pathways, the two key modes ofclearance of accumulated proteins in the endoplasmic reticulum,providing binding of retained proteins by the ER chaperone BiP (whichfacilitates translocation to proteolytic compartments). The presentinvention characterizes the involved mechanisms of intraceptor actionfor the subcellular consequences of KDEL (SEQ ID NO:1)-mediated proteinsequestration, and further provides opportunities for improvingtherapies for corneal angiogenesis or other disorders.

The present invention provides that intraceptors induce the unfoldedprotein response, upregulating p5OATFα, spliced XBP-1, BiP, and CHOP. Inpreferred embodiments, the intraceptors of the present invention induceapoptosis of vascular endothelial cells both in vitro and in viva, andthat the induced apoptosis is not rescued by external VEGF due tointraceptor-induced UPR by both intraceptors and downregulation ofVEGFR-2 and physiologic heterodimers by Flt24K (SEQ ID NO:6). Thepresent invention former provides that inhibition of UPR-activatedproteolytic mechanisms permits release of VEGF and VEGFR-2 from KDEL(SEQ ID NO:1) sequestration and hence decreases but not abolishesintraceptor-induced apoptosis of cells, as CHOP is also elevated byintraceptors. The present invention also provides that theubiquitin-proteasome pathway is the principal route for clearance ofintraceptor-retained proteins, as removal of KDEL (SEQ ID NO:1) fromPDI, an ER chaperone, has recently been described to release its targetprotein, procollagen 1, from ubiquitin-proteasome degradation (Ko & Kay,2004, Exp Cell Res 295:25-35). In one preferred embodiment, the presentinvention provides that disruption of normal heterodimer formation byFlt24K downregulates Ets-1, MMP-1, phosphorylation of FAK, and DNAsynthesis.

ERAD (endoplasmic reticulum associated degradation) remains poorlycharacterized. It is generally believed that retained proteins arepredominantly cleared by the proteasome or the lysosome (Ko & Kay, 2004,Exp Cell Res. 295:25-35; Hirsch et al., 2004, Biochim Biopghys Acta,1695:208-16; Kaufman, 2002, J Clin Invest 110: 1389-98; Werner et al.,1996, Proc Natl Acad Sci 93:13979-801). Mannosidase-1 has been proposedas art enzyme In a pathway of degradation within the ER itself formisfolded glycoproteins (Kaufman, 2002, J Clin Invest 110:1389-98); bothVEGF and VEGFR-2 are typically glycosylated. The present inventionprovides that the intraceptor activity includes proteasome or lysosomedegradation of VEGF or VEGFR-2. Alternatively, the present inventionprovides an inhibition of mannosidase-1 with kifunensine (Hosokawa etal., 2003, J Biol Chem. 278:26287-94; Mancini et al., 2003, J Biol Chem.278:46985-905) to demonstrate a pathway of degradation of VEGF orVEGFR-2 within, the ER itself. The present Invention also provides anexpression of a plasmid expressing a mutant ubiquitin (ubiquitin-K48R),which stops polyubiquitination and protects target proteins from theproteasome (Carter et al., 2004, J. Biol Chem, 279: 52835-9).

The present invention also provides that the mechanism ofintraceptor-mediated UPR activity may not lie entirely with ERAD or withCHOP induction. An early event in UPR is general downregulation oftranslation, mediated by PERK activation, which phosphorylateseokaryotic translation initiation factor 2 (elF2α), which attenuatesprotein synthesis in all eukaryotic cells (Ko & Kay, 2004, Exp Cell Res.295: 25-35). There is no inhibitor of elF2α or PERK. The presentinvention further provides studies in PERK null mice or mice defectivein elF2α phosphorylation (Seheuoer et al., 2001, Mol Cell, 7:1165-76) todemonstrate the effects of intraceptor-mediated UPR.

The present invention also provides that alternative markers ofphysiologic FLT/VEGFR-2 heterodimer function can include vinculinassembly and FAK phosphorylation. Vinculin assembly can be measured inHMECs after VEGF incubation for 30 minutes and 60 minutes is detected byindirect immunofluorescence assessing for viuculin localization to areasof focal adhesion. FAK phosphorylation induced by VEGF incubation for 30or 60 minutes is assessed, by immunoprecipitation of cell lysate usingantibody to phosphotyrosine, followed by western blotting using anantibody to p125^(FAK).

The present invention further provides that alternatives to apoptosisassessment include electron microscopy, TUNEL staining, staining withacridine orange/ethidium bromide, or ELISA for single-stranded DNA. Thepresent invention also provides that an alternative to measuring effectsof proteasome inhibition is evaluation of aggresome formation involvingintraceptor-sequestered target proteins. Aggresomes are pericentriolarstructures that accumulate ubiquitinated undegraded proteins mat aremisfolded. This can be performed using immunogold labeling andtransmission electron microscopy (Johnson et al., 1998, J Cell Biol143:1883-98).

Drug delivery of potential, intraocular therapies against angiogenesisis bedeviled by potential lack of sustainability. Topical plasmids donot cross the corneal epithelial barrier, and repeated intrastromalinjections are not clinically appealing. Gene therapy to date has alsorelied on use of viral vectors for long-term effect. These methods areplagued by virus-induced inflammation, potentiality for viralreplication and infectiousness, and possible induction of oncogenesis.

The present invention provides an alternative delivery approach, thecorneal electroporation. The present invention further provides thatreplication-deficient adenoviruses and lentiviruses are other potentiallong-term vectors but have disadvantages outlined above,Adeno-associated viruses are another alternative as well. Other methodsfor sustained delivery include use of integrase plasmids and use of theCre-lox system.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theattention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1 Specific Methods Cell Cultures

Except where indicated, the cell cultures were from ATCC, Manassas, Va.

HMECs (CDC) were grown in RPMI1640 medium supplemented with 10% fetalbovine serum (FBS) and 20 μg/mL bovine brain extract. Cells weremaintained at 37° C. in 5% CO₂, or on 1% gelatin-coated plates inmodified Kaighn's F 12K medium (ATCC) with 10% FBS, 0.03 mg/mlendothelial cell growth supplement (Sigma), and 50 U/ml heparin (Sigma).

Corneal epithelial cells (CRL-11, 515) were grown on culture-platesprecoated with 0.01 mg/ml fibronectin (Sigma), 0.01 mg bovine serum,albumin (BSA) (Sigma), and 0.03 mg/ml bovine collagen type I (Vitrogen100; Cohesion, Palo Alto, Calif.) in keratinocyte-serum free medium(ATCC) with 5 ng/ml human recombinant endothelial growth factor (EGF;Gibco, Carlsbad, Calif.), 0.05 mg/ml bovine pituitary extract (Gibco),0.005 mg/ml insulin (Sigma), and 500 ng/ml hydrocortisone (Sigma). Afterpassage 3, cells were used for experiments at approximately 30%confluence,

A375 cells were cultured in RPMI medium with 10% FBS, 100 units/mLpenicillin, and 100 μg/mL streptomycin.

LNCaP cells were cultured In RPMI medium with 10% FBS, 100 units/mLpenicillin, and 100 μg/mL streptomycin.

KG 1 cells were maintained in suspension cultures of 20% FBS and IMDM.

Hypoxia

Cells grown in 12 or 24 well culture plates were serum-starved for 12hours, then placed in a Coy Hypoxia Chamber (Coy, Grass take, Mich.)programmed for 5% oxygen-5% carbon dioxide-90% nitrogen, which studieshave shown are optimal for inducing VEGF without impairing cellviability (Namiki et al., 1995, J Biol Chem. 270:31189-95; Nomura et al.1995, 270:28316-24). Cell culture experiments were performed intriplicate.

Vector Construction

Separate vectors were constructed containing domains 2 and 3 or 2, 3,and 4 with the ER retention signal tag linked to the end of bothsequences. cDNAs encoding FLT domains 2-3 (Flt23) and domains 2, 3, 4(Flt24) (see FIG. 1) were amplified from a corneal cDNA library (OpenBiosystems). Human Rt-1 cDNA was used, as template DNA for PCR reactions(Open Biosystems). Primers were designed for the attachment of theretention signal tag to the truncated receptor sequences. Primers flt2-3(forward) (5′-TAG GAT CCA TGG ATA CAG GTA GAC CTT TCG TAG AG-3′ (SEQ IDNO:9) and flt2-3 (reverse) (5′-TAG AAT TCT ATT ACA GCT CGT CCT TTT TTCGAT GTT TCA CAG TGA-3′ (SEQ ID NO:10)) were used to amplify flt2-3/KDEL(SEQ ID NO:3). Primers flt2-3 (forward) and flt2-4 (reverse) (5′ TAG AATTCT ATT ACA GCT GGT CCT TGG CCT TTT CGT AAA TCT GG-3′ (SEQ ID NO:11))were used to amplify flt2-4/KDEL (SEQ ID NO:5). Both products weredigested with EcoRI/BamHI and cloned into a pCMV vector (Stratagene, LaJolla, Calif.), creating pCMV.Flt.23K and pCMV.Flt24K. The pCMV vectorscontaining the modified Flt-1 clones were transfected into competent E.coli cells, and selected for using kanamycin antibiotics.

Model/Inducing, Labeling, and Quantitation of Corneal Neovascularization

As previously described (Ambati et al., 20002, Arch Ophthalmol 120:1063-68), topical proparacaine and 2 μL of 0.15 M NaOH were applied toone cornea of each mouse. The corneal and limbal epithelia were removedusing a Tooke corneal knife (Arista Surgical Supply, New York, N.Y.) ina rotary motion parallel to the limbus. Erythromycin ophthalmic ointmentwas instilled immediately following epithelial denudation.

Immunohistochemical staining for vascular endothelial cells wasperformed on corneal flat mounts by a masked investigator. Fresh corneaswere dissected, rinsed in PBS for 30 minutes, and fixed in 100% acetone(Sigma) far 20 minutes. After washing in PBS, nonspecific binding wasblocked with 0.1 M PBS, 2% albumin (Sigma) for 1 hour at roomtemperature. Incubation with FITC-coupled monoclonal rat anti-mouse CB31antibody (BD Pharmingen) at a concentration of 1:500 in 0.1 M PBS, 2%albumin at 4° C. overnight was followed by subsequent washes in PBS atroom temperature. Corneas were mounted with an antifading agent(Gelmount; Biomeda, Inc; San Francisco, Calif.) and visualized with afluorescent microscope.

Images of corneal vasculature were captured using a CD-330charge-coupled device (CCD) camera attached to a fluorescent microscope(Proia et al., 1988, Lab Invest 58:473-79). The images were analyzedusing LSM-5 Image Examiner. (Zeiss; Germany), resolved at 624×480pixels, and converted to tagged information file format (TIFF) files.Neovascularization was quantified by setting a threshold level offluorescence above which only vessels were captured. The entire mountedcornea was analysed in masked fashion to minimize bias. The totalcorneal area was outlined using the innermost vessel of the Hmbal (rimof the cornea) arcade as the border. The total area ofneovascularization was then normalized to the total corneal area.

Transfection of Corneal Epithelial Cells

Corneal epithelial cells at 30% confluence were incubated withpCMV.Flt23K or pCMV.flt24K and transfection reagent (siPORT; Ambion,Austin, Tex.). Forty-eight hours after transfection, cells were placedin hypoxic conditions (5% O₂) in a hypoxia chamber (Coy LaboratoryProducts, Inc.). Three transfections were made per experiment.Nontransfected cells and cells transfected with empty pCMV vector servedas control cultures. The former were placed in hypoxia 48 hours afterreaching 30% confluence, although the latter were placed in hypoxia 48hours after transfection. It is contemplated that tire intraceptors willinteract with VEGF as shown in FIG. 2.

Immunoprecipitation

Cell lysate was diluted to 1 μg/μl total cell protein with PBS, of which1 mg lysate was then combined with indicated antibodies and incubatedovernight in 4 degrees. After 3 hours incubation at 4° C. in 20 μlwashed protein G agarose bead slurry (Upstate Cell Signaling,Charlottesville, Va.), followed by microcentrifuge pulsing, agarosebeads were recovered, washed 3 times in PBS, resuspended in 30 μl 2×Laemmli sample buffer, boiled for 5 minutes, and recovered again viamicrocentrifuge pulsing to yield supernatant for use in SDS-PAGE andimmunoblot analysis.

Quantification of Corneal Neovascularization by RT-PCR

After cell harvest, RNA was isolated using Qiagen RNA isolation kits.cDNA was made using 1 μg RNA using reverse transcriptase and oligo dTprimers; printers were synthesized by IDT after provision of appropriatesequences. Target RNA was amplified in 50 μL reaction mixture in athermal cycler (Eppendorf) for 35 cycles. Amplified products were run ina 2% agarose gel.

Corneal Intrastromal Injection

Effective transfection of plasmid delivery to the cornea has beendescribed previously (Stechschulte et al., 2001, Invest Ophthalmol VisSci 42:1975-79). A 30 gauge needle was used to nick the corneal stroma;a 33 gauge needle on a Hamilton syringe was passed through the nick tothe center and used to inject 1.2 μg plasmid in 2 μL of solution (or 2μL of PBS).

Harvest for ELISA

Culture medium or corneas harvested for ELISA were placed. In 60 μllysis buffet (20 nM imidazole hydrochloride, 10 mM potassium chloride, 1mM magnesium, chloride, 10 mM EGTA, 1% Triton X-100, 10 mM sodiumfluoride, 1 mM sodium molybdate, and 1 mM EDTA (ph 6.8)), supplementedwith protease inhibitor (Sigma), followed by homogenization. The lysatewas cleared of debris by centrifugation at 14,000 rpm for 15 minutes (4°C.), and the supernatant was collected. Total protein was determinedwith a Bradford protein assay (Bio-Rad).

VEGF-ELISA

VEGF was determined by a commercially available ELISA kit (R&D Systems)that recognizes the unbound 164-amino acid splice variant of mouse VEGF.The assay was performed according to the manufacturer's instructions.

Leukocyte Counts

Two days after corneal injury, corneas were embedded in optimal cuttingtemperature compound, frozen in liquid nitrogen, and cut into 7 μm thicksections After fixation with ice-cold acetone and blocking with normalgoat serum, sections were stained with monoclonal rat anti-mouse CD-45(leukocyte common antigen; BD PharMingen), followed by3,3-diaminobenzidine (DABE)-conjugated anti-rat IgG. Cells werevisualised by light microscopy and counted in a masked fashion at ×40.Eight consecutive serial sections were studied.

Western Blot

Corneal cell and matrix was harvested and placed in 150 μl RIPA buffer(Tris-HCL, NaCl, NP-40, Na-deoxycholate, and protease inhibitors).Immediately afterward, tissue samples were sonicated on ice four timesat 15-second intervals, each at level-7 intensity. After centrifugationssamples were loaded onto a 1.0% SDS-polyacrylamide gel, transferred, andprobed for VEGF protein. Membranes were blocked for 1 hour at roomtemperature with 5% milk in PBST, followed by overnight incubations at4° C in a concentration of 1:1000 VEGF primary antibody (BD PharMingen),which detects unbound VEGF. The appropriate secondary antibodyconcentration of 1:5000 (BD PharMingen) was used to incubate themembrane for 2 hours at room temperature, after which the membrane waswashed in PBST and developed on film using a chemiluminescence kit(ECL).

Statistics

Data analysis was performed on computer (Excel and SPSS for Windows).Statistical significance was assessed with Student's t-Test. Data areexpressed as the mean±SEM.

Xenografts

As described (Horton et al., 1999, Cane Res 59:4064-8), athymic 6week-old male BALB/C nu/nu mice were housed in pathogen-free conditionsin laminar flow boxes; mice were imunosuppressed by irradiation with 3Gy. One day later, one million A375 cells in 0.25 mL of culture mediumwere injected in upper back subcutaneously. Volumes were measured usinga caliper and calculated with the formula L×W×H×0.52 (Tomayko &Reynolds, 1989, Cancer Chemother Pharmacol, 24:148-54). At harvest,mouse and tumor weight were measured to control for mouse size.

Intradermal Tumor Angiogenesis

5 days after implantation, skin encompassing the rumor site was excisedand spread onto filter paper. Sections at 10× magnification wereassessed for total number of vessels; the mean value of vessels intissue from sham PBS-injected mice was subtracted from control andtreated mice to better determine the treatment efficacy (Wedge et al.,2002, Cancer Res. 62:4645-55).

Adhesion

Melanoma cells labeled with calcein AM were preincubated with blockingantibodies, then stimulated by VEGF. They were transferred to wellscoated with bone sialoprotein. At 1 hour, the wells were washed withDMEM by inversion, and adherent cells were quantitated by a fluorescentplate reader.

Cell Migration

Transfected cells are grown in normal medium then detached with trypsin.Cells are seeded in inner chambers of a 24-well transwellfibronectin-coated plate with 8-micron pores. In outer chambers, normalmedium serves as chemoattractant. Cells are incubated for 4-6 hours, thetop surface of the transwell is wiped clean, and the bottom surfacestained and examined by light microscopy. Cells on the underside of theplate are quantified in 10 random, fields at 200×.

Survival

Survival of cells was tested using trypan blue exclusion assay. Trypanblue was used as a marker of cell death since dead cells are incapableof excluding this dye. For each set of cells, a light microscope wasused to count the number of live and dead cells, with the minimum totalnumber of 100 cells per set. Survival was measured as the ratio of livecells to total cells counted.

Apoptosis

For in vitro experiments, cells were collected, centrifuged, andresuspended in 1× binding buffer (10 mM Hepes/NaOH, 140 mM NaCL, 2.5 mMcalcium chloride), supplemented with annexin V-FITC (BB PharMingen).After 15 minutes incubation in the dark, an excess of 1× binding bufferwas added to a final volume of 0.5 mL. Cells were analysed using flowcytometry.

Caspase assay was performed after lysing cells in caspase lysis buffetand centrifuging at 8000 g for 5 minutes. One hundred mg of protein, wasadded to the caspase reaction buffer and 100 μM of the peptideAc-DEVD-pNA. (Biomol Plymouth, Pa.). This was incubated at 37.degree,for 4 hours and then read at 405 nm. wavelength on a spectrophotometer.Reaction buffer and peptide without lysates served as control.

For in vivo experiments, corneas were fixed in formalin and sectionedinto 10 micron sections after paraffin-embedding. Sections were mountedonto slides, incubated overnight at 55° C, then deparafinized. Proteinwas digested with proteinase K at room temperature. After 4 washes indistilled water, endogenous peroxidase was quenched with 2% hydrogen,peroxide at room temperature and sections were washed twice in PBS.Labeling of 4′-OH fragmented DNA ends was performed with an in situapoptosis detection kit (ApopTag, Gaithersburg, Md.) followingmanufacturer's instructions. Detection of labeled ends was done with kitsupplied anti-digoxigenin-peroxidase antibody and development of DABsubstrate.

Proliferation

MTT, a tetrazolium salt, is cleaved into a blue-colored product byactive mitochondrial dehydrogenases, upregulated in proliferating cells.By colorimetrically measuring MTT before and after administration tocells, cellular metabolism is assessed. Using ELISA reader at 570 nm,proliferation is calculated as a percentage increase in absorbancecompared with empty media.

DNA Synthesis

10,000 HMECs were transfected with plasmids (1.2 μg plasmid in 2 μL)directing intraceptor expression or control plasmids at 30% confluence.The cells were preincubated for 24 hours, then incubated with or withoutVEGF 10 ng/mL and 1.0 μCi [methyl ³H] thymidine (Amersham) for 24 hours.Thymidine incorporation was measured by liquid scintigraphy.

Example 2 Intraceptors Suppress VEGF Upregulation & CornealNeovascularization in Human Corneal Epithelial (HCE) Cells

A clinically relevant model of corneal neovascularization was previouslyestablished. Sodium hydroxide epithelial denudation followed bymechanical scraping consistently induces 360 degrees ofneovascularization, a process driven by VEGF and its induced leukocyterecruitment (Ambati et al., 2002, Arch Ophthalmol. 120:1063-68; Amano etal., 1998, Invest Ophthalmol Vis Sci, 18-22). Angiostatin and geneticablation of chemokine receptors CCR2 or CCR5 can partially inhibitcorneal neovascularization in this model (Ambati et al., 2002, ArchOphthalmol. 120:1063-68; Ambati et al., 2003, Invest Ophthalmol Vis Sci,44:590-93; Ambati et al., 2003, Cornea 22:465-467).

pCMV.Flt23K and pCMV.Flt24K were generated as described in Example 1.

HCE cells were transfected at 30% confluency with pCMV.Flt23K,pCMV.Flt24K, or empty pCMV in order to confirm that VEGF is theangiogenic stimulator from hypoxia-conditioned corneal medium. 24 hoursafter transfection, the HCE cells were subjected to hypoxia and VEGFassays.

The Flt23K and Flt24K intraceptors were found to suppresshypoxia-induced VEGF secretion in HCE cells (FIG. 3A). In controlhypoxic HCE cells (experiments done in triplicate), VEGF expression inculture medium, increased from 286.8 pg/mL to 516.6 pg/mL (80.1%increase) after 17 hours of exposure to 5% oxygen and 457.6 pg/mL after24 hours (59.6% increase over baseline). Relative to the control,pCMV.Flt23K suppressed VEGF upregulation at 17 hours (VEGF increasedfrom 248.7 pg/mL to 389.6 pg/mL, (56.6% increase) (p=0.05)) but not at24 hours (VEGF level of 397.9 pg/mL, 59.9% above baseline (p=0.211)).Relative to the control, pCMV.Flt24K suppressed VEGF upregulation at 17hours (VEGF increased from 315.3 pg/mL to 398.6 pg/mL (26.4% decrease)(p=0.03)) and at 24 hours (VEGF level of 372.2 pg/mL, 18.0% abovebaseline) (p=0.04). The HCE cells transfected with both pCMV.Flt23K andpCMV.Flt24K had most suppression: baseline VEGF of 352.8 pg/mL rose to363.2 pg/mL at 17 hours (2.9% increase) (p=0.01) and was 361.1 pg/mL(2.4% increase over baseline) at 24 hours (p=0.02). The loss ofsuppression with Flt23K at 24 hours but not Flt24K may be due to thepresumed role of domain 4 in dimerization, which may enhance VEGFendoplasmic entrapment. While free VEGF was not detected in cell lysateby Western blotting (FIG. 3B), VEGF secretion (measured in the culturesupernatant) was not completely suppressed; this likely indicates thatintracellular sequestration is largely effective, while pre-formedstores of VEGF may have been released into extracellular space inresponse to hypoxia or by apoptotic or necrotic cells that release VEGF.

In summary, it has been found that VEGF secretion increases overbaseline in control, cells by 80% at 17 hours and by 60% at 24 hours.However, Flt23K suppresses the increase at 17 hours by 30% (p=0.05) butdoes not suppress the increase at 24 hours, and Flt24K suppresses theincrease at 17 hours by 73% (p=0.03) and suppresses the increase at 24 hby 70% (p=0.04).

Example 3 Intraceptors Inhibit VEGF, Leukocyte Infiltration &Neovascularization Due to Corneal Injury

To test the activity of VEGF intraceptors in vivo on cornealangiogenesis, and on two key angiogenesis-mediating elements; VEGFexpression, and leukocyte infiltration, PBS or plasmids expressingintraceptors were intrastromally injected, using a 33 gauge Hamiltonmicrosyringe needle. Two days later, corneal injury was performed, andone week later, corneas were harvested to quantify neovascularization.VEGF and leukocyte counts were assayed 2 days after injury. Tocharacterize the dose-response effects of intraceptors, 0.5, 1, 2, or 4pg of the respective plasmids were delivered into mouse cornea forinhibition and regression experimented and neovascularization, cornealVEGF levels, and leukocyte, infiltration were measured.

FIG. 4A shows the data. In mice (n=7 per subgroup), the mean percentagearea±SEM of corneal neocascularization 1 week after corneal injury was57.7±6.9% when injected with sham PBS or saline 2 days prior to injury,58.7±7.7% in mice injected with empty pCMV (p>0.05), 19.5±6.4% in miceinjected with pCMV.Flt23K (p=0.001), and 30.3±7.4% in mice injected withpCMV.Flt24K (FIG. 4A).

Two days after injury, corneal VEGF concentration was 1595.8±102.9 pg/μgof total protein in mice injected with PBS, 1.5.18.6±65.8 pg/μg in miceinjected with empty pCMV vector, 952.2±186.0 pg/μg in mice injected withpCMV.Flt23K p=0.009), and 1119.5±152.1 pg/μg in mice injected withpCMV.Flt24K (p=0.042) (FIG. 4B). Leukocyte counts per section were288.0±26.9 in mice injected, with PBS, 280.0±27.2 in mice injected withpCMV vector, 158.6±27.0 in mice injected with pCMV.Flt23K (p<0.001), and206.5±27.4 in mice injected with pCMV.Flt24K (p<0.0001) (FIG. 4C).

In summary, it has found that Flt23K suppresses injury-induced cornealneovascularization by 66%, corneal VEGF expression by 37%, and cornealleukocytes infiltration by 47% (p<0.01 for all). Flt24K suppressesinjury-induced corneal neovascularization by 49%, corneal VEGFexpression by 37%, and corneal leukocyte infiltration by 23% (p<0.05 forall). The studies also showed no significant differences between Flt23Kand Flt24K on corneal angiogenesis, VEGF expression and leukocyteinfiltration.

Example 4 Intraceptor Administration Regresses CornealNeovascularization

To determine whether intraceptors can regress cornealneovascularization, plasmids were delivered via corneal intrastromalinjection 7 days after corneal injury. This time point was selected asnaked plasmid expression has previously been shown to persist for 10days and corneal neovascularization begins to plateau approximately 8days after injury (Stechschulte et al., 2001, Invest Ophthalmol Vis Sci42:1975-79).

Corneal injury was performed on the mice, and PBS, empty pCMV vector,pCMV.Flt23K, or pCMV.Flt24K were intrastronmally injected into theircorneas 7 days later. Photographs of the corneas were taken at 7 days,14 days, and 17 days after injury (data not shown). The data showed thatpCMV.Flt23K and pCMV.Flt24K can regress corneal neovascularization. Themean percentage area of corneal neovascularization 14 days after cornealinjury and 10 days after intrastromal injection was 40.4±2.7% in miceinjected with empty pCMV, 23.4±7.0% in mice injected with pCMV.Flt23K(p=0.001), and 19.3±6.1% in mice injected with pCMV.Flt24K (p<0.001)(data, not shown). These data demonstrated that the intraceptorsregressed corneal, neovascularization when administered after thecorneal injury, and specifically demonstrate that the neovascularizationat 14 days in the intraceptor-treated groups is less than the control at14 days and at 7 days.

Example 5 Intraceptors Sequester VEGF, Suppress VEGF Secretion Inducedby Hypoxia in Melanoma Cells and Flt24K Binds VEGFR-2

A375 human, malignant melanoma cells (HMMCs) were known to express onlyVEGFR-2 and not Flt. Therefore, any results using HMMCs are notconfounded by intrinsic Flt/VEGF complexes or Flt/VEGFR-2 heterodimers.

HMMCs transfected with pCMV.Flt23K. or pCMV.Flt24K were subjected tohypoxia 48 hours after transfection. Two days later, the cellularfraction was immunoprecipitated with anti-FLT antibody (epitope specificfor domains 2 and 3; Santa Cruz, Calif.) and subsequently underwentWestern blotting with an anti-VEGF or an anti-VEGFR-2 antibody. A 50 kDaband (consistent with a VEGF homodimer interacting with, theintraceptor) was detected in cells transfected with pCMV.Flt23K orpCMV.Flt24K (FIG. 5A). A 30 kDa band was present in cells transfectedwith pCMV.Flt24K but not pCMV.Flt23K (FIG. 5B). This indicates thatFlt24K can hensrodimerize with both VEGF and VEGFR-2. The isolated bandIs consistent with a VEGFR-2 fragment (as it is significantly smallerthan the known molecular weight of VEGFR-2 (200-220 kDa)).

Intraceptor suppression of VEGF secretion induced by hypoxia in HMMCswas also determined. After 72 hours of hypoxia, extracellular VEGF inthe medium of control HMMCs was 4977.74±297.60 pg/ml, while that in HMMCtransfected with pCMV.Flt23 one day prior to hypoxia induction was2323.88±150.75 pg/ml (experiments done in triplicate, p<0.001; nosignificant difference in baseline VEGF levels or cell number) (FIG. 6).

Example 6 Intraceptor Inhibit Melanoma Xenograft Growth in Nude Mice

A375 xenografts were placed subcutaneously into BALB/c athymic (nude)mice. One million HMMC cells grown in vitro were injected subcutaneouslyin the upper dorsum of nude mice. Weekly injections of pCMV.Flt24K orcontrol (saline) into the xenograft were administered, and tumor growthwas measured biweekly. Five mice were used in each group. Flt24K (SEQ IDNO:6) was observed to dramatically suppress the growth of melanoma inthis model by 83.8% over 6 weeks (FIG. 7) (p<0.001; F=19.76).Representative photographs are also shown (FIGS. 7B and C).

In further experiments, plasmids directing Flt23K (SEQ ID NO:4) orFlt24K (SEQ ID NO:6) or both are injected into the xenograft at the timeof tumor placement or 7 days later, and tumor growth is evaluated,weekly for 6 weeks. Controls include empty pCMV vector and PBS. Tumorangiogenesis is assessed by microscopy to count vessels. Additionally,A375 cells are transfected with pCMV.Flt23K or pCMV.Flt24K or both(controls included PBS and empty pCMV vector). They are grown in aserum-free medium in 5% hypoxia for 48 hours, and viable cells arecounted by trypan blue exclusion, using a hemacytometer. Apoptosis isassessed by using TUNEL staining or caspase 3 levels. VEGFR-2 expressionis assessed by flow cytometry, and VEGF secretion is also assessed byELISA. Immunohistochemistry is used to determine colocalization ofintraceptors with VEGF and VEGFR-2 in the endoplasmic reticulum toverify success of this approach in melanoma cells. Adhesion is assessedby adherence to bone sialoprotein, and migration by transwell plates. Toassess whether the presumed autocrine loops are intracellular, thecontrol groups includes cells treated with neutralizing anti-VEGFmonoclonal antibodies or extracellular soluble VEGFR-1.

Example 7

Alkali-Mechanical Trauma Does Not Induce Significant Lymphangiogenesis

To determine whether lymphangiogenesis is a significant component in themodel of corneal neovascularization induced by alkali-mechanical trauma,vessels were labeled with rabbit anti-mouse LVYE-1, a lymphatic marker,10 days after corneal injury. A minimal lymphangiogenesis (<10% ofcorneal area compared to −60% for hemangiogenesis) was found (data notshown).

Example 8

Intraceptors Induce the Formation of Spliced XBP-1 In Vitro and In Vivoand Induce CHOP

The unfolded protein response (UPR) induces conversion, of theconstitutive 30 kD form of X-box binding protein 1 (XBP-1) throughalternative splicing to its active 55 kDa form, which inducesendoplasmic reticulum associated degradation of sequestered proteins. Todetermine whether intraceptors induce the UPR, HMECs were transfectedwith pCMV.Flt23K or pCMV.Flt24K. Controls included empty pCMV and PBS.Twenty-four hours after transfection, cells were placed in 5% hypoxiafor 6 hours, and cell lysates were analysed by Western blotting withanti-XBP-1 antibody (Chemicon). The 55 kDa spliced variant of XBP-1 wasupregulated in cells transfected with intraceptors, consistent with aninduction of the UPR (FIG. 8).

For the in vivo study, corneal injury was performed, and 10 days later,empty pCMV, pCMV.Flt23K, or pCMV.Flt24K was intrastromally injected.Mouse corneas were harvested 2 days later and Western, blotting wasperformed for XBP-1 (experiments in triplicate; FIG. 9). Theintraceptors promoted the conversion of the inactive form of XBP-1 toits active form (FIG. 9). These data suggested that accumulation ofsequestered intraceptor-VEGF complexes in the endoplasmic reticulummight lead to endoplasmic reticulum overload, triggering the unfoldedprotein response (UPR), which by itself or in combination withdownregulation of VEGF could cause apoptosis of endothelial cells.

In addition to XBP-1 splicing, the UPR induces CHOP, which is associatedwith apoptosis. To test whether intraceptor delivery would elevate CHOP,HMECs were transfected with pCMV.Flt23K and pCMV.Flt24K, in vitro andRT-PCR for CHOP expression was performed 24 hours later (experiments intriplicate). It was found that intraceptors induce elevation of CHOPlevels (FIG. 10).

In vivo expression of the intraceptors in the cornea is cornea byR.T-PCR and Western blot.

To determine whether intraceptors are more successful, at inhibitingcorneal angiogenesis than extracellular VEGF blockade, plasmidsexpressing intraceptors or insert slow-release EVA copolymer pelletscontaining 10 pg anti-VEGF antibody (a dose sufficient to blockextracellular VEGF) are delivered into a corneal pocket One day later,corneal neovascularization is induced by combined mechanical-alkalitrauma. Corneal VEGF levels are assessed 2 days later and cornealneovascularization 1 week later.

To determine whether intraceptors induce apoptosis and trigger the UPR,plasmids expressing pCMV.Flt23K or pCMV.Flt24K are transfected in HMECs.Empty pCMV vector and reagent alone are transfected as controls. After 3days of incubation, cell proliferation, is assessed using the MTT assaywith ELISA, survival by trypan blue exclusion, and apoptosis by annexinV staining and caspase-3 ELISA. After 1 day of incubation, UPRactivation is assessed by assessing levels of p5OATF6α, spliced XBP-1,BiF, and CHOP by Western blot and RT-PCR. To further determine whetherapoptosis is due to the ER stress-induced UPR or to mere down regulationof VEGF, VEGF (10 ng/mL) is added to the culture medium of cells, asthis would be expected to rescue cells from the latter but not theformer. To confirm that UPR activation is specifically directed towardsretained VEGF or VEGFRb-2, immunoprecipitation is performed withanti-BiP antibody (Santa Cruz) and Western blotting with antibodies toVEGF or VEGFR-2. These experiments are carried out both in normoxic and5% hypoxic-conditions.

To further characterize whether apoptosis is due to UPR-inducedproteolysis of VEGF or to CHOP induction, plasmids expressingpCMV.Flt23K or Flt24IC are transfected in HMEC, with the same controlsas above. One day later, UPR-activated proteolytic mechanisms areinhibited using 10 pM of MG132 (potent inhibitor of both,ubiquitin-proteasome pathway and lysosomes), lactacystin (inhibitor ofproteasomes) and chioroquine (inhibitor of lysosomes). The UPR-activatedproteolytic mechanisms are known to be the predominant mechanism ofprotein, digestion in ERAD (Ko & Kay, 2004: Exp Cell Res. 295:25-35).

The effect of proteasome inhibition is measured by assessingaccumulation of ubiquitinated VEGF or VEGFR-2 throughimmunoprecipitation with anti-VEGF or and VEGFR-2 antibody followed byWestern blot with anti-ubiquitin antibody. The effect of lysosomeinhibition, is measured by assessing accumulation of VEGF or VEGFR-2 inthe Golgi complex by double immunostaining with anti-Golgi 58k proteinantibody and anti-VEGF or VEGFR-2 antibody (Ko & Kay, 2004, Exp CellRes. 295:25-35). To determine whether proteolytic blockade may allowescape of sequestered proteins from UPR into the medium or cell surface,VEGF secretion and VEGFR-2 levels are measured by ELISA and flowcytometry, respectively, after 12 hours.

To assess the role of CHOP in inducing apoptosis in this study, aplasmid expressing an siRNA against CHOP is developed and co-transfectinto HMECs along with plasmids expressing intraceptors. Apoptosis isassessed 24 hours later using above-noted methods. To further definewhether apoptosis is due primarily to endoplasmic reticular stress,procaspase 12 expression is assessed by ELISA, as caspase 12 activationis specific to apoptosis induced by endoplasmic reticular stress(Kaufman, 2002, J Clin Invest. 110; 1389-98; Schroder & Kaufman, 2005,Mutat Res. 560:29-63). These experiments are carried out both innormoxic and 5% hypoxic conditions.

To determine whether intraceptors suppress cellular events normallymediated by physiologic FLT/VEGFR-2 heterodimers, plasmids expressingpCMV.Flt24K are transfected in HMEC, with controls as above. One dayafter transfection the cells are incubated with VEGF (10 ng/mL). After 4hours of incubation, cells are harvested for RT-PCR analysis of Ets-1and MMP-1. DNA synthesis is assessed by incubating cells with VEGF andtritiated-methyl thymidine and measuring incorporation of the latterafter 24 hours by liquid scintigraphy.

To determine whether intraceptors induce apoptosis in vivo, based on themethods described above, corneal neovascularization is induced bycombined mechanical-alkali trauma. pCMV.Flt23K and pCMV.Flt24K aredelivered via corneal intrastromal injection 7 days after cornealinjury; controls are PBS and empty pCMV vector that were delivered aswell. At 10, 15, and 20 days after injury, apoptosis is assessed byperforming dual labeling with CD31 and annexin V immunostaining oncorneas. Ghost vessels are identified by using dual-immunolabeling withantibodies to collagen IV and CD31 (a ghost vessel would be positive forcollagen IV but not CD31, while the corneal stroma, where vesselsgenerally are present, normally does not have collagen IV (Baluk et al.,2003, Am J Path. 163; 1801-15).

To determine the effects of intraceptors on corneal neovascularendothelium, corneal neovessel endothelial (CD31⁻CD45⁻) cells areisolated by magnetic cell sorting of neovascularized corneal cellsuspensions yielded by collagenase digestion to analyze intracellalarevents (unfolded protein response markers, heterodimerization-inducedevents, apoptosis-related markers) specifically in these cells. Mousechoroidal macrophages are previously isolated using magnetic cellsorting Yoshida et al., 1997, Mol Cell Biol. 17; 4015-23). MMP-1, XBP-1,p50ATF6, and CHOP expression is assessed by Western blotting inuninjured corneas, then, at 4, 8, 12, and 16 days after injury (asexpected, these markers of intraceptor activity precede apoptosis) inboth whole corneas by Western blotting and from corneal neovesselendothelial cells by RT-PCR.

What is claimed is:
 1. An intraceptor that interacts with VEGF and/or aVEGFR, said intraceptor comprising a chimeric polypeptide comprising:(a) a portion of SEQ ID NO: 13 operatively linked to a Signal retentionpeptide, or (b) a polypeptide having at least 80% sequence identity with30 consecutive amino acids of SEQ ID NO: 13 operatively linked to asignal retention peptide.
 2. The intraceptor of claim 1, wherein saidportion of SEQ ID NO:13 comprises amino acids 1-305 of SEQ ID NO:6. 3.The intraceptor of claim 1, wherein said portion of SEQ ID NO:13comprises amino acids 1-211 of SEQ ID NO:4.
 4. The intraceptor of claim1, wherein said VEGFR is selected from the group consisting of VEGFR-1,VEGFR-2 and VEGFR-3.
 5. The intraceptor of claim 1, wherein said VEGFRis VEGFR-2.
 6. The intraceptor of claim 1, wherein said signal retentionpeptide prevents secretion of the portion of SEQ ID NO: 13 operativelylinked to the retention peptide.
 7. The intraceptor of claim 1, whereinsaid signal retention peptide is an endoplasmic reticulum signalretention peptide.
 8. The intraceptor of claim 7, wherein saidendoplasmic reticulum signal retention peptide is selected from thegroup consisting of SEQ ID NO: 1, 7, or
 8. 9. The intraceptor of claim7, wherein said endoplasmic reticulum signal retention peptide is SEQ IDNO:1.
 10. A method for treating an angiogenesis-related condition in apatient, said method comprising: contacting a cell of the patientinvolved in the angiogenesis-related condition with an intraceptorcomprising a chimeric polypeptide comprising a portion of SEQ ID NO: 13operatively linked to a signal retention peptide, or a polypeptidehaving at least 80% sequence identity with 30 consecutive amino acids ofSEQ ID NO: 13 operatively linked to a signal retention peptide, whereinsaid contact decreases activity of VEGF and/or a VEGFR.
 11. The methodof claim 10, wherein the intraceptor is selected from the groupconsisting of: a) a polypeptide as defined in SEQ ID NO:4; b) apolypeptide as defined in SEQ ID NO:6; and c) a polypeptide having atleast 80% sequence identity with the polypeptide of a) through b) above.12. The method of claim 10, wherein said portion of SEQ ID NO: 13comprises amino acids 1-305 of SEQ ID NO:6.
 13. The method of claim 10,wherein said portion of SEQ ID NO:13 comprises amino acids 1-211 of SEQID NO:4.
 14. The method of claim 10, wherein said signal retentionpeptide is an endoplasmic reticulum signal retention peptide selectedfrom the group consisting of SEQ ID NO: 1, 7, or
 8. 15. The method ofclaim 14, wherein said endoplasmic reticulum signal retention peptide isSEQ ID NO:
 1. 16. The method of claim 10, wherein the VEGFR is VEGFR-2.17. The method of claim 10, wherein said angiogenesis-related conditionis selected from the group consisting of inflammation, stroke,hemangioma, solid tumors, leukemias, lymphomas, myelomas, metastasis,telangiectasia psoriasis sclerodenna, pyogenic granuloma, myocardialangiogenesis, plaque neovascularization, coronary collaterals, ischemiclimb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma,diabetic retinopathy, retrolental fibroplasia, arthritis, diabeticneovascularization, macular degeneration, wound healing, peptic ulcer,fractures, keloids, vasculogenesis, hematopoiesis, ovulation,menstruation, placentation, polycystic ovary syndrome, dysfunctionaluterine bleeding, endometrial hyperplasia and carcinoma, endometriosis,failed implantation and subnormal foetal growth, myometrial fibroids(uterine leiomyomas) and adeuomyosis, ovarian hyperstimulation syndrome,ovarian carcinoma, melanoma, venous ulcers, acne, rosacea, warts,eczema, neurofibromatosis, tuberous sclerosis, and chronic inflammatorydisease.
 18. The method of claim 10, wherein the disease or condition isselected from the group consisting of: melanoma, diabetic retinopathy,and macular degeneration.
 19. The method of claim 18, wherein thedisease or condition is macular degeneration.